JP2023001803A - cross-linked collagen - Google Patents

Abstract

To provide a cross-linked collagen having sufficient strength and high safety.SOLUTION: A cross-linked collagen in a first embodiment includes: a unit derived from collagen; a unit derived from an amino group-containing polymer compound comprised of a polyamino acid, an aminated polysaccharide, and a polyallylamine; and a unit derived from an anhydrous carboxylic acid. A cross-linked collagen in a second embodiment is identical to the cross-linked collagen in the first embodiment except that the collagen is obtained by crosslinking using a reaction product between the amino group-containing polymer compound and the anhydrous carboxylic acid. The cross-linked collagen gel can be suitably used as a cell culture substrate or the like.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to crosslinked collagen that can be widely used as a medical material, etc., and a crosslinker that can be used to produce crosslinked collagen and the like.

Medical materials used in contact with living bodies or cells include cell culture substrates such as scaffolds for cell growth, sutures, medical instruments such as wound dressings, artificial blood vessels, artificial heart valves, artificial ligaments, Artificial tissue materials such as artificial tendons, artificial bones, and artificial cartilage.

Collagen is widely used as such a medical material. Collagen is one of the proteins that mainly constitute the dermis, ligaments, tendons, bones, cartilage, etc. of vertebrates. Collagen is characterized by having a primary structure in which glycine residues are repeated every three residues, and is classified into types such as type I to type XI depending on the amino acid sequence, peptide length, presence or absence of fibril formation, and the like.
Collagen has excellent adhesion and interaction with cells, low immunogenicity, excellent bioavailability and biocompatibility, and can be molded into various shapes such as sheets, fabrics, fibers, and tubes. and is suitable as a medical material. In particular, collagen is in great demand as a material for cell culture substrates.

Biological tissues are composed of cells and an extracellular matrix composed of various types of molecules such as collagen, proteoglycan, glycosaminoglycan, and hyaluronic acid. When a living tissue is damaged, the extracellular matrix is lost together with the cells, so artificial scaffolding materials are implanted in the injured site to replace the lost extracellular matrix. This promotes cell differentiation and proliferation, speeding damage repair. Also, cells are proliferated in a container having an artificial scaffold formed on the bottom surface, and the resulting tissue is transplanted to the injured site.
The scaffold material secures a space for tissue regeneration and plays a role of helping regeneration of damaged tissue while maintaining the shape of the regenerated tissue. In order to play this role, the scaffold material should have good adhesion to cells, biocompatibility or biocompatibility, low immunogenicity, and the ability of the scaffold that is no longer needed after burying to be biocompatible. It is required to be easily absorbed and strong enough not to break even when the body is moved.

Similarly, medical instruments and artificial tissues are required to have biocompatibility and low immunogenicity because they are used in contact with living bodies. In addition, in order not to apply mechanical stress to the living body, it is necessary to have appropriate stretchability and strength that does not break even when stretched, that is, elasticity.

Collagen does not adversely affect the living body, but its strength is insufficient. Therefore, attempts have been made to cross-link collagen to improve its strength.
For example, Patent Document 1 discloses that collagen is solubilized, and an aldehyde-based cross-linking agent such as glutaraldehyde, an epoxy-based cross-linking agent such as ethylene glycol diglycidyl ether (PGDE), 1-ethyl-3-(3-dimethyl Collagen gels crosslinked with carbodiimide crosslinkers such as aminopropyl)carbodiimide (EDC) and isocyanate crosslinkers such as hexamethylene diisocyanate are disclosed.
Patent Document 2 also discloses collagen gels crosslinked with crosslinkers such as glutaraldehyde, divinylsulfone, epoxide, carbodiimide, imidazole, N-hydroxysuccinimide, thiol-derivatized polyethylene glycol.

However, it has been pointed out that these conventional crosslinked collagens are cytotoxic due to the crosslinker. Therefore, there is a demand for crosslinked collagen that has sufficient strength and is highly safe.

JP 2012-130386 Special Table No. 2010-528046

An object of the present invention is to provide crosslinked collagen that has sufficient strength and is highly safe. Another object of the present invention is to provide a safe cross-linking agent that can be used to produce cross-linked collagen having sufficient strength.

In order to solve the above problems, the present inventors have conducted repeated studies, and have found that units derived from collagen, units derived from amino group-containing polymer compounds composed of polyamino acids, aminated polysaccharides, and polyallylamine, and units derived from carboxylic anhydride. It has been found that the crosslinked collagen containing units has excellent mechanical strength, no or almost no cytotoxicity, and high safety.

The present invention has been completed based on the above findings, and provides the following [1] to [9].
[1] A crosslinked collagen comprising units derived from collagen, units derived from an amino group-containing polymer compound composed of polyamino acid, aminated polysaccharide, and polyallylamine, and units derived from carboxylic anhydride.
[2] The crosslinked collagen according to [1], which is obtained by crosslinking collagen using a reaction product of the amino group-containing polymer compound and carboxylic anhydride.
[3] The crosslinked collagen of [1] or [2], which is a gel.
[4] The reaction product of the amino group-containing polymer compound and the carboxylic anhydride is obtained by carboxylating 50 to 100 mol% of the amino groups of the amino group-containing polymer compound with the carboxylic anhydride [1 ] to [3].
[5] The crosslinked collagen according to any one of [1] to [4], wherein the amino group-containing polymer compound is polylysine.
[6] The crosslinked collagen according to any one of [1] to [5], wherein the collagen is derived from fish.
[7] A cell culture substrate comprising the crosslinked collagen according to any one of [1] to [6].
[8] A cross-linking agent containing a reaction product of an amino group-containing polymer compound comprising a polyamino acid, an aminated polysaccharide, and a polyallylamine, and a carboxylic anhydride.
[9] The cross-linking agent of [8], wherein 50 to 100 mol% of the amino groups of the amino group-containing polymer compound are carboxylated with carboxylic anhydride.

Unlike the conventional crosslinked collagen taught in Patent Documents 1 and 2, the crosslinked collagen of the present invention is highly safe because it uses a crosslinker that has no or almost no cytotoxicity. Therefore, it can be suitably used as a medical material that is used in contact with living organisms, biological tissues, or cells.
In addition, the material to be used in contact with the living body needs to have appropriate stretchability so as not to give stress to the living body, and also needs to have strength so that it does not break even if it is stretched to some extent. The conventional crosslinked collagen taught in Patent Documents 1 and 2 is brittle and lacks practicality in terms of mechanical strength. Prepare. Therefore, it is useful as a medical material in terms of strength as well.

The present invention will be described in detail below.
The crosslinked collagen of the present invention is a crosslinked collagen containing units derived from collagen, units derived from an amino group-containing polymer compound composed of polyamino acid, aminated polysaccharide, and polyallylamine, and units derived from carboxylic anhydride.
Among them, the crosslinked collagen of the present invention may be a crosslinked collagen obtained by crosslinking collagen using a reaction product of the amino group-containing polymer compound and carboxylic anhydride.

Collagen Collagen has been isolated from living organisms and identified in a plurality of types that differ in terms of amino acid sequence, peptide length, presence or absence of fibril formation, etc. Any type of collagen can be used in the present invention. Examples thereof include collagen type I, II, III, IV, V, VI, VII, VIII, IX, X, and XI.
Collagen may be derived from mammals such as cows, pigs, horses, chickens and humans, from birds, or from fish such as tuna and cod. Examples of human-derived collagen include those expressed from human placenta cDNA and those produced by culturing human fibroblasts.
Since collagen derived from mammals and birds has low solubility in water, it is not possible to obtain a collagen gel containing collagen at a high concentration. difficult. Moreover, when bovine- or porcine-derived collagen is used as a raw material, there is a possibility that pathogens of mad cow disease and swine fever are brought into the crosslinked collagen. Sites where such pathogens may exist are not usually used as collagen raw materials, but should be avoided in the case of medical applications. Therefore, fish-derived collagen is preferred.
One type of collagen may be used, or two or more types having different origins or molecular weight distributions may be used.

Polyamino acids Polyamino acids include polymers of basic amino acids such as polylysine, polyarginine, and polyhistidine.
Among them, polylysine is preferable from the viewpoint of good supply stability. Among polylysines, ε-poly-L-lysine has been used as a food preservative and α-poly-L-lysine has been used as a cell adhesive, therefore their safety has been confirmed. It can be preferably used at points. ε-poly-D-lysine, α-poly-D-lysine can also be used.
Polylysine having a number average molecular weight in the range of 100 to 100,000 produced by Streptomyces bacteria is commercially available. Among them, those having a number average molecular weight of 1,000 to 20,000, particularly 1,000 to 10,000 are preferred. In terms of degree of polymerization, those with a number average degree of polymerization of 15 to 35 and those with a degree of polymerization of 20 or less are also produced and can be used. The number average molecular weight and number average degree of polymerization are values measured by SDS-polyacrylamide gel electrophoresis.

Aminated polysaccharides Aminated polysaccharides are those in which all or part of the hydroxyl groups of polysaccharides have been aminated.
Polysaccharides include β-glucan (laminaran, curdlan, cellulose, etc.), α-glucan (pullulan, amylose, glycogen, amylopectin, dextran, dextrin, etc.), chitosan, xanthan gum, karaya gum, gellan gum, locust bean gum, tamarind gum. , tragacanth gum, guar gum, acacia gum, tamarind seed gum, tara gum, gum arabic, alginic acid, sodium alginate, pectin, galactomannan, agar, etc.

Polyallylamine Polyallylamine preferably has a weight average molecular weight of 1,000 to 1,000,000, more preferably 1,000 to 100,000, more preferably 1,000 to 20,000. Commercially available products such as allylamine polymer (PAA-03; Nitto Boseki Co., Ltd.) are also available.

Among amino group-containing polymer compounds, polyamino acids are preferred.
One or more amino group-containing polymer compounds can be used.

（式(1)中、R1及びR2は、それぞれ独立して、水素原子、又はC1～C4のアルキル基である。R3及びR4は、それぞれ独立して、水素原子、又はC1～C4のアルキル基である。）
中でも、R1及びR2が水素原子であり、R3及びR4が水素原子以外の上記官能基である化合物が好ましく、R1、R2、及びR3が水素原子であり、R4が水素原子以外の上記官能基である化合物がより好ましく、R1、R2、R3、及びR4が水素原子である化合物（コハク酸無水物）が特に好ましい。 Carboxylic anhydride Carboxylic anhydride is not particularly limited, and dicarboxylic anhydride, tetracarboxylic dianhydride and the like can be used. Among them, dicarboxylic anhydrides are preferred, and examples of dicarboxylic anhydrides include succinic anhydride represented by the following formula (1) and derivatives thereof.

(In formula (1), R1 and R2 are each independently a hydrogen atom or a C1-C4 alkyl group. R3 and R4 are each independently a hydrogen atom or a C1-C4 alkyl group. is.)
Among them, preferred are compounds in which R1 and R2 are hydrogen atoms, and R3 and R4 are the above functional groups other than hydrogen atoms, and R1, R2, and R3 are hydrogen atoms, and R4 is the above functional groups other than hydrogen atoms. Certain compounds are more preferred, particularly those in which R1, R2, R3, and R4 are hydrogen atoms (succinic anhydrides).

（式(2)中、R1及びR2は、それぞれ独立して、水素原子、又はC1～C4のアルキル基である。R3及びR4は、それぞれ独立して、水素原子、又はC1～C4のアルキル基である。）
中でも、R1及びR2が水素原子であり、R3及びR4が水素原子以外の上記官能基である化合物が好ましく、R1、R2、及びR3が水素原子であり、R4が水素原子以外の上記官能基である化合物がより好ましく、R1、R2、R3、及びR4が水素原子である化合物（ジグリコール酸無水物）が特に好ましい。 Diglycolic anhydride represented by the following formula (2) or a derivative thereof can also be used.

(In formula (2), R1 and R2 are each independently a hydrogen atom or a C1-C4 alkyl group. R3 and R4 are each independently a hydrogen atom or a C1-C4 alkyl group. is.)
Among them, preferred are compounds in which R1 and R2 are hydrogen atoms, and R3 and R4 are the above functional groups other than hydrogen atoms, and R1, R2, and R3 are hydrogen atoms, and R4 is the above functional groups other than hydrogen atoms. Certain compounds are more preferred, particularly those in which R1, R2, R3, and R4 are hydrogen atoms (diglycolic anhydride).

（式（3）中、R1及びR2は、それぞれ独立して、水素原子若しくはC1～C4のアルキル基であるか、又はR1及びR2が一体となって、C1～C6のアルカン－ジイル基を形成している。R3及びR4は、それぞれ独立して、水素原子若しくはC1～C4のアルキル基であるか、又はR3及びR4が一体となって、C1～C6のアルカン－ジイル基を形成している。R5及びR6は、それぞれ独立して、水素原子若しくはC1～C4のアルキル基であるか、又はR5及びR6が一体となって、C1～C6のアルカン－ジイル基を形成している。） Glutaric anhydride represented by the following formula (3) or a derivative thereof can also be used.

(In formula (3), R1 and R2 are each independently a hydrogen atom or a C1-C4 alkyl group, or R1 and R2 together form a C1-C6 alkane-diyl group. R3 and R4 are each independently a hydrogen atom or a C1-C4 alkyl group, or R3 and R4 together form a C1-C6 alkane-diyl group (R5 and R6 are each independently a hydrogen atom or a C1-C4 alkyl group, or R5 and R6 together form a C1-C6 alkane-diyl group.)

The C1-C4 alkyl groups can be, for example, C1-C3, especially C1-C2 alkyl groups. Examples of alkane-diyl groups include C1-C3 alkane-1,1-diyl groups and C4-C6 alkylene groups. The combination of R1 and R2, the combination of R3 and R4, and the combination of R5 and R6 are hydrogen atom and hydrogen atom, hydrogen atom and methyl group, hydrogen atom and ethyl group, methyl group and methyl group, methyl group and ethyl group, and ethyl. and ethyl groups. Examples of C1-C3 alkane-1,1-diyl groups include methane-1,1-diyl groups and ethane-1,1-diyl groups. The C4-C6 alkylene group includes a tetramethylene group (butane-1,4-diyl group) and a pentamethylene group (pentane-1,5-diyl group).
Among them, R1 and R2 are the above functional groups other than hydrogen atoms, and R3, R4, R5, and R6 are hydrogen atoms, or R3 and R4 are the above functional groups other than hydrogen atoms, and R1, R2, and R5 , R6 are hydrogen atoms.

3,3-ジメチルグルタル酸無水物（DMGA）

3-メチルグルタル酸無水物（MGA）

2,2-ジメチルグルタル酸無水物

3,3-テトラメチレングルタル酸無水物

3-オキサスピロ[5,5]ウンデカン-2,4-ジオン(3,3-ペンタメチレングルタル酸無水物）

Among the compounds of formula (3), the following compounds are preferred.
Glutaric anhydride (GA)

3,3-dimethylglutaric anhydride (DMGA)

3-methylglutaric anhydride (MGA)

2,2-dimethylglutaric anhydride

3,3-tetramethyleneglutaric anhydride

3-oxaspiro[5,5]undecane-2,4-dione (3,3-pentamethyleneglutaric anhydride)

Among the dicarboxylic anhydrides, compounds of formula (1) are preferred.
One or more carboxylic anhydrides can be used.

Method for Producing Crosslinked Collagen The cross-linking agent used for cross-linking collagen is a compound obtained by reacting the amino group-containing polymer compound with carboxylic anhydride to carboxylate some or all of the amino groups of the amino group-containing polymer compound. be.
In this cross-linking agent, it is preferable that 50% or more, especially 60% or more, especially 65% or more of the amino groups of the amino group-containing polymer compound are carboxylated. Further, 100% or less of the amino groups of the amino group-containing polymer compound may be carboxylated, but 80% or less or 70% or less may be carboxylated. Within this range, the mechanical strength of the crosslinked collagen is sufficient.

In cross-linking collagen with the above-mentioned cross-linking agent, collagen may be dissolved in water and reacted with the cross-linking agent. The collagen may be dissolved in water and reacted with the cross-linking agent at the same time, or the collagen may be dissolved in water and then reacted with the cross-linking agent. Commercial products of collagen dissolved in water, such as collagen BM (Nitta Gelatin Co.), can also be used.
Collagen may be dissolved in water using an acid. When an acid is used, the pH of the aqueous solution should be adjusted to about 2-3. It can also be solubilized using an enzyme such as collagenase.

The cross-linking reaction can be performed by mixing the solubilized collagen and the cross-linking agent. In the case of acid solubilization, the pH of the aqueous solution may be adjusted to 6-8, especially 7 after cross-linking. The cross-linking reaction may be carried out for 5-60 minutes.
In addition, the solubilization and cross-linking reactions may be performed at a temperature at which collagen is not denatured, for example, 4 to 37°C, especially 20 to 37°C.
The amount of the cross-linking agent used can be 0.1 parts by weight or more, especially 0.5 parts by weight or more, especially 1 part by weight or more per 1 part by weight of collagen. Within this range, a gel having a sufficient crosslink density can be obtained, and the crosslinked collagen has sufficient mechanical strength. The amount of the cross-linking agent used can be 100 parts by weight or less, especially 75 parts by weight or less, especially 50 parts by weight or less per 1 part by weight of collagen. It can also be 25 parts by weight or less, or 10 parts by weight or less. Within this range, the cross-linking agent will not be excessive with respect to the reaction site of collagen.

Properties of Crosslinked Collagen The crosslinked collagen of the present invention can be, for example, a crosslinked collagen gel. Gels include hydrogels containing water and xerogels (porous gel sponges) obtained by drying them. Drying methods include freeze-drying and vacuum-drying. In addition, non-gelled crosslinked collagen may be used.
In addition, crosslinked collagen can be formed into various shapes depending on the application, and examples thereof include forms such as sheets or films, fabrics such as non-woven fabrics, fibers, threads, tubes, lumps, powders, and granules.

Uses The crosslinked collagen of the present invention is particularly suitable for use as a medical material. For example, scaffolds for cell culture (scaffolds for cell proliferation), cell culture substrates such as scaffolds for in vivo tissue regeneration; artificial blood vessels, artificial heart valves, artificial joints, artificial skin, artificial ligaments, artificial tendons, artificial bones , artificial tissues such as artificial cartilage; surgical sutures, wound dressings, dental materials, tissue adhesives, heart pacemakers, stents and other medical devices embedded in the body; catheters, contact lenses, drug Suitable as a material for medical instruments used in contact with a living body such as base materials for broadcasting and endoscopes; medical instruments used in contact with living body separation materials such as hemodialysis membranes and blood storage packs. can be used for

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these.

(1) Production of crosslinked collagen gel
(1-1) Synthesis of cross-linking agent
PLL-SA(65)
Succinic anhydride is added to the amino group of ε-poly-L-lysine (molecular weight 4000, JNC Co., Ltd.) so that it becomes 15 to 50 mol%, reacted at 50 ° C. for 1 hour, and carboxylated polylysine Synthesized. The amount of carboxyl groups introduced was 65% with respect to the side chain amino groups of ε-poly-L-lysine. The resulting reactant is abbreviated as PLL-SA (65).

PLL-DMGA(65)
The synthesis method of PLL-SA (65) was the same except that 3,3-dimethylglutaric anhydride (DMGA) was used instead of succinic anhydride. The amount of carboxyl groups introduced was 65% with respect to the side chain amino groups of ε-poly-L-lysine. The resulting reaction is abbreviated as PLL-DMGA (65).

(1-2) Production of crosslinked collagen gel
Example 1
1.0 g of tuna collagen powder (Hiragane Sangyo Co., Ltd., HK Collagen) was added to 9 g of water, and the mixture was stirred well while being heated to 60°C. . Dissolution of tuna collagen was confirmed by the fact that the solution became transparent. The pH was confirmed with pH test paper. Next, equal amounts of a 10% by weight aqueous solution of PLL-SA (65) and the tuna collagen solution were mixed and stirred at 25° C. for 10 minutes. Further, a 5 mol/L sodium hydroxide aqueous solution was added dropwise to adjust the pH to 7.
400 μL of the resulting solution was poured into a circular silicon mold with a diameter of 2 cm, dried overnight at room temperature, and molded into a sheet. The collagen sheet was removed from the silicone mold, placed in an autoclave bag, and vacuum-dried at 50° C. for 3 days, and further vacuum-dried at 160° C. for 3 hours for cross-linking and drying to obtain a dry cross-linked collagen sheet. The resulting dry crosslinked collagen sheet is a crosslinked collagen xerogel (porous crosslinked collagen sponge).

Example 2
A dry crosslinked collagen sheet was obtained in the same manner as in Example 1, except that PLL-DMGA (65) was used instead of PLL-SA (65).

Example 3
In Example 1, porcine collagen solution (3 mg/mL, Cell Matrix Type IA, Nitta Gelatin Co., Ltd.) was used instead of the tuna collagen solution prepared using 1.0 g of tuna collagen powder (Hiragane Sangyo Co., Ltd., HK Collagen). A dry crosslinked collagen sheet was obtained in the same manner as in Example 1, except that 10 g was used. The crosslinked collagen concentration in the hydrogel before drying is 0.15% by weight.

Comparative example 1
A dry crosslinked collagen sheet was obtained in the same manner as in Example 1, except that glutaraldehyde (GA) was used instead of PLL-SA(65).

Comparative example 2
A dried crosslinked collagen sheet was prepared in the same manner as in Example 1, except that 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used instead of PLL-SA (65). got

Comparative example 3
A dry crosslinked collagen sheet was obtained in the same manner as in Example 1, except that ε-poly-L-lysine (molecular weight: 4000, JNC Corporation) was used instead of PLL-SA(65). rice field.

Comparative example 4
A dried collagen sheet was obtained in the same manner as in Example 1, except that the cross-linking reaction with PLL-SA(65) was not performed.
That is, 1.0 g of tuna collagen powder (Hiragane Sangyo Co., Ltd., HK Collagen) is added to 19 g of water, stirred well while being heated to 60°C, and 5 mol/L hydrochloric acid is added dropwise to adjust the pH to about 2 to dissolve the tuna collagen. let me Dissolution of tuna collagen was confirmed by the fact that the solution became transparent. The pH was confirmed with pH test paper. Then, a 5 mol/L sodium hydroxide aqueous solution was added dropwise to adjust the pH to 7. This resulted in a hydrogel of uncrosslinked collagen.
400 μL of the resulting solution was injected into a circular silicon mold with a diameter of 2 cm, dried overnight at room temperature, and molded into a sheet. The collagen sheet was taken out from the silicon mold, placed in an autoclave bag, vacuum-dried at 50° C. for 3 days, and further vacuum-dried at 160° C. for 3 hours to obtain a dried collagen sheet. The dried collagen sheet obtained is a collagen xerogel (porous collagen sponge).

(2) Evaluation
(2-1) Cytotoxicity The collagen sheet prepared in each of the above examples was sterilized by soaking in 70% ethanol overnight, washed with PBS three times, and cultured (Nacalai Tesque Co., Ltd., Dulbecco's Modified Eagle Medium). It was immersed in glucose (containing 10% fetal bovine serum) and allowed to swell overnight. 50,000 mesenchymal stem cells (MSCs) were seeded on the swollen sheet, cultured at 37°C for 1 day in the presence of 5% CO ₂ , and the state of the cells was observed and evaluated according to the following criteria.
O: Cells uniformly adhered to the collagen sheet.
Δ: Cells adhered to the collagen sheet but were not uniform.
x: The cells were in a floating state without adhering to the collagen sheet.

(2-2) Elastic modulus (tensile test)
The elastic modulus of the collagen sheet produced in each example above was measured using Autograph AGS-J manufactured by Shimadzu Corporation. The initial load was 0.5 N, the tensile speed was 1 mm/min, and the test was conducted in 5 series (n=5). The shape of the test piece was JIS dumbbell type 7, the size of the sample, and the distance between gauge points (distance between grips) were within the following ranges.

・Example 1
Width 1.97-2.37mm, thickness 0.094-0.108mm; gauge length 14.59-15mm
・Example 2
Width 2.2mm, thickness 0.096mm; gauge length 14.8mm
・Comparative example 1
Width 1.66-2.06mm, thickness 0.033-0.049mm; gauge length 13.32-14.97mm
・Comparative example 2
Width 1.38-2.23mm, thickness 0.035-0.043mm; gauge length 13.82-15.06mm
・Comparative example 4
Width 1.89-2.15mm, thickness 0.038-0.045mm; gauge length 14.59-15.43mm

※比較例6は、ブタコラーゲンをEDC/NHS（N-ヒドロキシスクシンイミド）で架橋したものであり、弾性率は、ｈｔｔｐｓ：／／ｍｉｅ－ｕ．ｒｅｐｏ．ｎｉｉ．ａｃ．ｊｐ／ から入手可能な論文「組織工学的人工血管のための高強度コラーゲン材料の開発」より引用した値である。 Table 1 shows the results.

* In Comparative Example 6, porcine collagen was crosslinked with EDC/NHS (N-hydroxysuccinimide), and the elastic modulus was obtained from https://mie-u. repo. nii. ac. The value is quoted from the article "Development of High-strength Collagen Material for Tissue-engineered Artificial Blood Vessels" available from jp/.

The crosslinked collagen gels of Examples 1 and 2 exhibited elastic moduli approximately 1.8 times and approximately 1.7 times higher than the non-crosslinked collagen gel of Comparative Example 4, respectively.
In addition, the crosslinked collagen gels of Comparative Examples 1 and 2 using GA or EDC, which are conventional crosslinking agents, had moderate cytotoxicity, but the crosslinked collagen gels of Examples 1 and 2 exhibited no cytotoxicity. didn't have it at all.

The cross-linked collagen gels of Comparative Examples 1 and 2 using GA and EDC as cross-linking agents have the same elastic modulus as the non-cross-linked collagen gel of Comparative Example 4, and polylysine was used as the cross-linking agent. The crosslinked collagen of Comparative Example 3 was very fragile and cracked after production, making it difficult to put into practical use.

The crosslinked collagen gel of Example 3 using porcine collagen had a low collagen concentration and was soft, so the elastic modulus could not be measured, but it can be used for applications where softness is desired. The hardness can also be improved by adjusting the ratio of polylysine and succinic anhydride in the cross-linking agent, the cross-linking rate of collagen, and the like.

The crosslinked collagen of the present invention is very useful as a medical material because it has both excellent strength and biocompatibility.

Claims (9)

A crosslinked collagen comprising units derived from collagen, units derived from an amino group-containing polymer compound comprising a polyamino acid, an aminated polysaccharide, and polyallylamine, and units derived from carboxylic anhydride. 2. The crosslinked collagen according to claim 1, wherein collagen is crosslinked using a reaction product of said amino group-containing polymer compound and carboxylic anhydride. 3. Crosslinked collagen according to claim 1 or 2, which is a gel. Claims 1 to 3, wherein the reaction product of said amino group-containing polymer compound and carboxylic anhydride is obtained by carboxylating 50 to 100 mol% of amino groups of said amino group-containing polymer compound with carboxylic anhydride. Crosslinked collagen according to any one of . The crosslinked collagen according to any one of claims 1 to 4, wherein the amino group-containing polymer compound is polylysine. The crosslinked collagen according to any one of claims 1 to 5, wherein the collagen is derived from fish. A cell culture substrate comprising the crosslinked collagen according to any one of claims 1 to 6. A cross-linking agent containing a reaction product of an amino group-containing polymer compound comprising a polyamino acid, an aminated polysaccharide, and a polyallylamine, and a carboxylic anhydride. 9. The cross-linking agent according to claim 8, wherein 50 to 100 mol % of amino groups in the amino group-containing polymer compound are carboxylated with carboxylic anhydride.