JP2002369878A - Material for internal regeneration of tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a material for repairing a tissue defect which is capable of rapidly healing the defect site of the tissue or organs without requiring extracorporeal cell culture in repairing and healing of the tissue defect part. SOLUTION: The internal regenerative material of tissue characterized in being composed of a cell adhesive active material (A) and a bioabsorptive material (B), is used. (A) is preferably a polypeptide (A1) which is synthesized by gene recombination microorganisms and has at least one piece (more preferably 3 to 50 pieces) of the minimum amino acid sequence indicating a cell adhesion signal within one molecule. The minimum amino acid sequence indicating the cell adhesion signal is preferably at least one kind of sequence selected from the group consisting of an RDG sequence, LDV sequence, REDV sequence, YIGSR sequence, PDSGR sequence, RIVVLPR sequence, LGTIPG sequence, RNIAEIIKDI sequence, IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a material for regenerating tissue in vivo. More specifically, muscle, skin tissue, bone, cartilage, blood vessels, nerves, tendons, ligaments,
Follicular tissue, mucosal tissue, periodontal tissue, dentin, bone marrow, retina,
The present invention relates to an implantable tissue regeneration material that can be applied to defective sites such as serosa, gastrointestinal tract, and fat, and to defective sites of organs such as lung, liver, pancreas, and kidney to promote regeneration of the tissues and organs. .

[0002]

2. Description of the Related Art A bioabsorbable polymer containing fibroblasts for promptly growing a living tissue has been proposed as a repair material for tissue defects by combining a bioabsorbable material and tissue cells. An artificial augmentation material containing fibroblasts in an alginate gel and a combination of chondroitin sulfate are also known (JP-A-6-3436).
No. 89). Furthermore, cultured artificial skin (Organogenesis (Orga)) comprising a combination of collagen sponge and cells.
(Genesis, Inc., Gunze Co., Ltd.) and cultured artificial skin in which cells are combined with polyglycolic acid (Advanced Tissue Science (Advanced Tissue Science)).
ue Science) has been commercialized, and a technique for regenerating pulmonary arteries by combining vascular constituent cells with a polyglycolic acid nonwoven fabric (Tokyo Women's Medical University: Toshiharu Shinoka, “BioClinica,” Vol. 15, No. 14 (2000) Year) 1123
P.).

[0003]

When a repair material is prepared by incorporating its own cells into a bioabsorbable material such as alginic acid, tissue cells are cut out from a site that is not defective.
It takes one to several weeks for the cells to grow and fill the entire repair material according to the size of the defective tissue, and there is a problem that it takes too much time from the defect to the completion of the treatment. An object of the present invention is to provide a tissue defect repair material capable of promptly treating a defect site of a tissue or an organ in repair and healing of a tissue defect portion without the need for extracellular cell culture in repair and healing of the tissue defect portion. is there.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventor has found that the use of a cell-adhesive substance and a bioabsorbable substance allows the substance to be quickly implanted from a non-defective tissue. And found that the cells rapidly proliferated and differentiated, and reached the present invention. That is, the feature of the tissue regeneration material of the present invention in the body is that it is composed of the cell adhesion active substance (A) and the bioabsorbable substance (B).

[0005]

DESCRIPTION OF THE PREFERRED EMBODIMENTS First, the cell adhesion active substance (A) will be described. (A) is a substance having cell adhesion activity, and a natural substance and an artificial substance can be used. Natural substances include glycoproteins present in the basement membrane (eg, laminin, entactin (nidogen), tenascin, agrin, osteonectin, osteocalcin, osteopontin, fibrin, fibrinogen, vitronectin, ancarin, bamin, thrombospondin, etc.), Proteoglycans (eg, aggrecan, perlecan, biglycan, decorin, fibromodulin, versican, durin, neurocan, brevican, lumican, serglycine, syndecan, CD4
4. Beta glycans, thrombomodulin, glypican, celebroglycan, NG2 proteoglycan, etc., glycoproteins present in cell membranes (eg, integrins, integrin superfamily, cadherin and cadherin superfamily, etc.), substances related to tight junctions (eg, Occludin, etc.).

As an artificial substance, for example, a polypeptide (A1) or the like which is synthesized by a genetically modified microorganism and has at least one minimal amino acid sequence in one molecule which represents a cell adhesion signal is used. Polypeptide (A
In 1), as the minimum amino acid sequence representing a cell adhesion signal, any one that functions as an adhesion signal can be used. For example, "Pathophysiology" Vol. 9, No. 7 (1990) at page 527.

[0007] Among them, RGD sequence, LDV sequence, REDV sequence (1), YIGSR sequence (2), PDSGR sequence (3) expressed by one letter of amino acid in that many types of cells adhere. , RYVVLPR sequence (4), LGTIPG sequence (5), RNIAEIIKD
I sequence (6), IKVAV sequence (7), LRE sequence, D
The GEA (8) sequence and the HAV sequence are preferred, and more preferably the RGD sequence, the YIGSR sequence (2),
DSGR sequence (3), LGTIPG sequence (5), IKV
AV sequence (7) and HAV sequence, particularly preferably RGD
Sequence, IKVAV sequence (7) and HAV sequence. The polypeptide (A1) may contain at least one sequence of these minimum amino acid sequences, and may contain a combination of two or more sequences.

It is necessary that at least one of the above-mentioned minimum amino acid sequences is contained in one molecule in the polypeptide (A1). When the minimum amino acid sequence is not contained, cell adhesion tends to be reduced, resulting in insufficient growth of cells on or in the present material. The content of the minimum amino acid sequence is 3 to 3 per molecule from the viewpoint of cell adhesion / proliferation.
The number is preferably 50, more preferably 4 to 30, and particularly preferably 5 to 20.

The number average molecular weight of the polypeptide (A1) is
In terms of low toxicity to cells and high adhesion performance,
It is preferably from 5,000 to 5,000,000, more preferably from 10,000 to 1,000,000, particularly preferably from 50,000 to 500,000. That is, the number average molecular weight of (A1) is preferably 5,000 or more, more preferably 10,000 or more, particularly preferably 50,000 or more, and preferably 5,000,000 or less, more preferably 1,5 or less. It is at most 100,000, particularly preferably at most 500,000. In addition,
The number average molecular weight of the polypeptide (A1) was determined by SDS-PA
It can be obtained by developing (A1) in water by GE method (Na dodecyl sulfate-polyacrylamide gel electrophoresis method) and comparing the migration distance with a standard substance.

The polypeptide (A1) has, besides the minimum amino acid sequence showing a cell adhesion signal, heat stability, structural stability, affinity with the bioabsorbable substance (B) and / or Amino acid sequence that improves the water solubility more appropriately, for example, amino acid sequence G derived from silk fibroin
It is preferable to have AGAGS (9) or the like, and to have at least two (preferably 3 to 50)
, More preferably 5 to 20).

As a preferred specific example of the polypeptide (A1), one molecule of Pronectin F (manufactured by genetically modified Escherichia coli, manufactured by Sanyo Kasei Kogyo Co., Ltd.) contains one cell adhesion signal RGD sequence and (GAGAGS) ₉ sequence (10) A polypeptide having a number average molecular weight of about 100,000 and a Pronectin F plus (produced by reacting Pronectin F with dimeraminoethyl chloride to make it water-soluble); a Pronectin L (genetically modified E. coli) Cell adhesion signal IKVAV sequence (7) and (GA
GAGS) polypeptide having a number average molecular weight of about 100,000, each having about 12 ₉ sequences (10) in one molecule.

Other artificial cell adhesive peptides available on the market include, for example, RGDS [a peptide comprising the RGDS sequence (11), a number average molecular weight of about 400,
GRGDS [peptide consisting of GRGDS sequence (12), number average molecular weight about 500,
Peptide Research Institute Co., Ltd.], RetroNectin
(Recombinant human fibronectin CH-296)
[Human fibronectin cell adhesion signal CS1
A peptide having one signal and one cell adhesion domain Type III and one heparin binding domain II, a number average molecular weight of about 60,000, manufactured by Takara Shuzo Co., Ltd.], and RGDS-Pr
protein A "The RGD sequence is replaced with protein A (I
gG binding domain), a number average molecular weight of about 30,000, manufactured by Takara Shuzo Co., Ltd.].

The polypeptide (A1) is produced by a genetically modified microorganism (for example, yeast, bacteria, Escherichia coli, etc.), and can be easily obtained by the method described in, for example, Japanese Patent Publication No. 3-502935. Can be Although it can be produced by chemical synthesis, it is preferable that it is synthesized by microorganisms because it is uniform and has excellent cell proliferation.

Next, the bioabsorbable substance (B) will be described. The bioabsorbable substance (B) can be used without limitation as long as it is embedded in the defective tissue and serves as a scaffold for the surrounding tissue cells to proliferate. For example, natural polymers, synthetic polymers, inorganic substances, and composites thereof are used. Examples of natural polymers include, for example, collagen, gelatin, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparin, elastin,
Chitin, chitosan, fibrin, alginic acid, starch, dextran, albumin, polyhydroxybutyric acid,
Examples include pectin, pectic acid, galactan, pullulan, agarose, gluten, fibroin, and derivatives thereof.

These derivatives include partially decomposed products thereof, oxides, alkylene oxide adducts, carboxymethylated products, crosslinked products and the like. As these partial degradation products, a partial degradation product by an enzyme (for example, collagenase or the like) or the like is used, and examples thereof include atelocollagen. As these oxides, oxides using an oxidizing agent (for example, hydrogen peroxide and the like) are used, and examples thereof include oxidized starch and the like.

As these alkylene oxide adducts, adducts of alkylene oxides having 2 to 6 carbon atoms (eg, ethylene oxide) are used, and examples thereof include hydroxyethylated starch. These carboxymethylated products include, for example, carboxymethylated starch and the like. As these crosslinked products,
A crosslinked product by a crosslinking agent (for example, glutaraldehyde or the like) or heat (for example, 100 to 200 ° C.) is used, and examples thereof include a thermally crosslinked product of collagen.

As the synthetic polymer, for example, a monomer selected from the group consisting of lactic acid, leucine, glycolic acid, ε-caprolactone, dioxanone, malic acid, lactide and glycolide is used as an essential monomer (co). Examples include a polymer (polyglycolic acid), a synthetic polypeptide not containing the minimum amino acid sequence representing a cell adhesion signal, and polycyanoacrylate. The number average molecular weight (based on gel permeation chromatography, hereinafter abbreviated as Mn) of these synthetic polymers is 1,
It is preferably from 000 to 5,000,000, more preferably from 5,000 to 1,000,000, particularly preferably from 10,000 to 500,000. That is, Mn of the synthetic polymer is preferably 1,000 or more, more preferably 5,000 or more, and particularly preferably 10,000 or more.
Or more, preferably 5,000,000 or less,
It is more preferably at most 1,000,000, particularly preferably at most 500,000.

The glass transition temperature of these synthetic polymers is preferably from -50 to 150 ° C, more preferably from -20 to 100 ° C, particularly preferably from -10 to 80 ° C. That is, the glass transition temperature (° C.) of the synthetic polymer is
-50 or more is preferable, -20 is particularly preferable, -10 is particularly preferable, and 150 or less is preferable,
It is more preferably 100 or less, particularly preferably 80 or less. These synthetic polymers can be easily obtained by an ordinary method.For example, polyglycolic acid is obtained by converting monomers such as lactic acid, glycolic acid and caprolactone to 150 to 150 mol in the presence of an inorganic solid acid catalyst such as acidic clay. Heat to 250 ° C,
A method of performing a dehydration reaction under reduced pressure for 10 to 150 hours, or a method of heating a cyclic monomer (for example, lactide or the like) to 150 to 250 ° C. in the presence of a catalyst such as octyltin and reacting for 10 to 150 hours. Obtainable.

The synthetic polypeptide is prepared by converting amino acid monomers to 50-250 amino acids in the presence of an inorganic solid acid catalyst such as acid clay.
It is obtained by heating to 1O <0> C and performing a dehydration reaction under reduced pressure for 1 to 150 hours. The polycyanoacrylate is obtained by reacting a cyanoacrylate monomer in air at a temperature of 10 to 100 ° C. for 1 to 150 hours at normal pressure.

As the inorganic substance, for example, calcium carbonate, calcium phosphate and the like are used. Examples of the calcium carbonate include light calcium carbonate and heavy calcium carbonate. Examples of the calcium phosphate include hydroxyapatite, tricalcium phosphate, and mixtures of these with other calcium phosphates (eg, monocalcium hydrogen phosphate and the like). Hydroxyapatite is Ca ₁₀ (PO ₄ ) ₆ (O
H) a basic calcium phosphate salt represented by _2,
It is synthesized using any of a wet method, a dry method, and a hydrothermal method such that the Ca / PO ₄ molar ratio is 1.67, and may be a commercially available reagent, or may be a porous apatite, granule, or the like. Apatite may be used.

Also, tricalcium phosphate is
It is represented by Ca ₃ (PO ₄ ) ₂ and is synthesized by a wet method or a dry method so that the molar ratio of Ca / PO ₄ is 1.50. Good. Further, as a mixture with other calcium phosphate, for example, monocalcium hydrogen phosphate, α
-A mixture of tricalcium phosphate (α-TCP) and calcium carbonate (CC). Although the method for producing these inorganic materials is not particularly specified, for example, a dry method, a hydrothermal method, a wet method, and an alkoxide method can be applied, and heat treatment may be performed as a post-treatment.

As these composites, composites composed of a natural polymer and a synthetic polymer are used. As the complex composed of a natural polymer and a synthetic polymer, a block and / or a graft polymer obtained by reacting a monomer constituting the synthetic polymer with the natural polymer is used. Starch and the like. The complex composed of a natural polymer and a synthetic polymer can be obtained, for example, by performing a polymerization reaction of monomers constituting the synthetic polymer in the presence of the natural polymer.

Among these bioabsorbable substances (B), from the viewpoint of material handling, bioabsorbable substances selected from the group consisting of the following bioabsorbable substances (1) to (4) Preferably, more preferably, the following (5) to (6)
Is a bioabsorbable substance selected from the group consisting of bioabsorbable substances represented by (1) Natural polymers Collagen, gelatin, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, fibrin, alginic acid, chitin, chitosan, fibroin, starch, pectin, pectic acid, and their partially decomposed products, oxides, alkylene oxides Adducts, carboxymethylated products and crosslinked products.

(2) Synthetic polymer (co) polycondensate containing a monomer selected from the group consisting of lactic acid, glycolic acid and ε-caprolactone as an essential constituent monomer, and a minimum amino acid exhibiting a cell adhesion signal Polypeptide having at least one sequence in one molecule (A
Synthetic polypeptides other than 1). (3) Inorganic substances Tricalcium phosphate, hydroxyapatite and calcium carbonate. (4) Complex composed of natural polymer and synthetic polymer A block and / or graft copolymer obtained by polymerizing lactic acid, glycolic acid and / or ε-caprolactone on a natural polymer.

(5) Natural polymers Collagen and gelatin. (6) Synthetic polymer A (co) polycondensate comprising a monomer selected from the group consisting of lactic acid, glycolic acid and caprolactone as an essential constituent monomer.

The shape of (B) is preferably powder, granule, fiber, cloth, tube, film, gel, sponge, block, etc., and more preferably powder, fiber, cloth. , Gels and sponges, particularly preferably powders, gels and sponges, most preferably gels and sponges. Shape is average particle size 0.1
Spherical, porous, or fibrous forms having an average cross-sectional diameter of 0.01 mm to 5 mm made of an aggregate of μm to 300 μm.

The term “powder” refers to an average particle diameter (for example,
A value indicated by an average value obtained by measuring the diameter of 10 particles, preferably 50 particles, under a microscope. The same applies hereinafter. ) 0.00
It means a shape of 1 μm to 1 cm (preferably 0.1 to 1000 μm). The term “granular” means a shape having a particle diameter of 1 to 5 cm (preferably 1 to 3 cm). The term “fibrous” refers to an average diameter (for example, a value indicated by an average value obtained by measuring the diameter of 10 sections, preferably 50 sections using a vernier caliper or a microscope, the same applies hereinafter).
1 cm (preferably 1 μm to 5 mm) and 1 cm to 1 length
m (preferably 1 cm to 10 cm).

The term "fabric" refers to a thickness (for example, a value indicated by an average value obtained by measuring the thickness of 10 places, preferably 50 places using a caliper or a microscope; the same applies hereinafter).
0.1 μm to 10 cm (preferably 1 μm to 5 cm) and an area of 0.1 to 1000 cm ² (preferably 1 to 100 cm)
2) means the shape of woven or non-woven fabric. In addition, the term “tubular” refers to an inner diameter (for example, the cross section is 1
A value indicated by an average value obtained by measuring the inner diameter of 0 places, preferably 50 places. The same applies hereinafter. ) 0.005 to 5 cm (preferably 0.01 cm to 1 cm) and an outer diameter (for example, a value indicated by an average value obtained by measuring the outer shape of 10 sections, preferably 50 sections using a vernier caliper or a microscope. 0.01 to 10 cm (preferably 0.05 to 2 c)
m), length 0.1 to 50 cm (preferably 0.5 to 10 cm)
cm).

[0029] The film shape means a thickness of 0.1 µm to 1 µm.
0 cm (preferably 1 μm to 5 cm) and area 0.1 to 1
000 cm ² (preferably 1 to 100 cm ² ). In addition, the size of the gel is 0.001 to 100c.
m3 (preferably 0.1 to 10 cm3) and a water content of 5 to 99
% By weight (preferably 20 to 95% by weight) non-flowable. In addition, the sponge shape is a size of 0.00
A value indicated by an average value of the average pore diameter (for example, the cross section is measured at 10 places, preferably 50 places using a microscope in a cross section) at 1 to 100 cm ³ (preferably 0.1 to 10 cm ³ ). ) Means a shape of 0.01 to 1000 μm (preferably 0.1 to 100 μm). Also,
The term "block-shaped" means a material having a size of 0.001 to 100 cm3 (preferably 0.1 to 10 cm3) other than the above (the same applies hereinafter).

The content of the cell adhesion active substance (A) is 0.00000 with respect to 100 parts by weight of the bioabsorbable substance (B).
The content is preferably 1 to 500 parts by weight, more preferably 0.0
001 to 50 parts by weight, particularly preferably 0.1 to 20 parts by weight. That is, the content (parts by weight) of (A) is
(B) It is preferably at least 0.00001, more preferably at least 0.0001, particularly preferably at least 0.1, and preferably at most 500, based on 100 parts by weight,
It is more preferably 50 or less, particularly preferably 20 or less. When the content of (A) is within this range, it becomes easy to achieve both the cell adhesion and proliferation properties and the physical properties (strength, flexibility, etc.) of the material, and the effect of promoting the healing of tissue defects can be more sufficiently exerted. There is a tendency.

The material of the present invention may contain a solvent (C) in addition to the cell adhesion active substance (A) and the bioabsorbable substance (B). The solvent (C) is not particularly limited as long as it can dissolve or disperse the cell adhesion active substance (A) and / or the bioabsorbable substance (B), but includes inorganic salts, amino acids, vitamins, organic acid salts, and alcohols. An aqueous solution containing 0.1 to 50% by weight (preferably 1 to 30% by weight) of a lipid, a saccharide, an acid and / or a base, and water can be used.

Examples of the inorganic salt include an alkali metal halide, an alkaline earth metal halide, an alkali metal carbonate,
Alkali metal sulfate, alkaline earth metal sulfate, transition metal sulfate, alkali metal nitrate, transition metal nitrate, alkali metal phosphate and alkali metal perchlorate can be used, for example,
Lithium chloride, lithium bromide, sodium chloride, potassium chloride, calcium chloride, calcium bromide, sodium carbonate, potassium hydrogen carbonate, sodium sulfate, potassium sulfate, potassium hydrogen sulfate, magnesium sulfate, calcium sulfate, copper sulfate, iron nitrate, nitric acid Examples include sodium, potassium nitrate, iron nitrate, sodium phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, lithium perchlorate, sodium perchlorate, and lithium perchlorate.

As the amino acid, an amino acid having 2 to 11 carbon atoms and the like can be used. For example, arginine, histidine,
Isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, valine,
Alanine, asparagine, aspartic acid, glutamic acid, proline, serine, glycine and the like. Examples of vitamins include choline, inositol, nicotinamide, glutamine, vitamin A and vitamin B1.
2 and the like.

As the organic acid salt, a salt of an organic acid having 1 to 6 carbon atoms can be used, and examples thereof include potassium oxalate and sodium acetate. As the alcohol, an alcohol having 1 to 4 carbon atoms can be used, and examples thereof include methanol, ethanol, isopropyl alcohol, and butanol. Examples of the lipid include fatty acid glycerides (for example, glycerides of fatty acids (C8 to C22) and the like), phospholipids (for example, phosphate esters of fatty acids (C8 to C22) and the like), glycolipids (for example, glycosphingolipids) And sulfolipids).

Examples of saccharides include monosaccharides (eg, glucose, erythrose, ribose, fructose, etc.),
Disaccharides (eg, lactose, sucrose, trehalose, maltose, etc.), oligosaccharides (eg, amylose,
Amylopectin) and amino sugars (eg, the amino acids). Examples of the acid include an inorganic acid and one having 1 carbon atom.
And organic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, formic acid, phenol and hexanedioic acid. As the base, an inorganic base and an organic base having 2 to 6 carbon atoms can be used, and examples thereof include sodium hydroxide, potassium hydroxide, ammonia, ethylamine, ethylenediamine, pyridine and triethylamine.
Examples of the water include distilled water, ion-exchanged water, tap water, and ion-exchanged distilled water.

Of these solvents (C), aqueous solutions containing inorganic salts, acids and / or bases, and water are preferred.
More preferably, it is an aqueous solution containing an inorganic salt and water, particularly preferably an aqueous solution containing an alkali metal halide, an alkali metal carbonate and / or an alkali metal phosphate. When the solvent (C) is used, the content of (C) is
For a total weight of 100 parts by weight of (A) and (B),
It is preferably 0.1 to 100,000 parts by weight, more preferably 1 to 10,000 parts by weight, particularly preferably 10 to 100 parts by weight.
5,000 parts by weight. That is, in this case, (C)
Is preferably 0.1 or more, more preferably 1 or more, particularly preferably 10 or more, based on 100 parts by weight of the total content of (A) and (B). 2,000 or less, more preferably 1 or less.
It is at most 5,000, particularly preferably at most 5,000.

When the solvent (C) is not used, (A)
(B) is distributed in a solid state on the surface and / or inside, and when a solvent (C) is used, (A) is dispersed in a solid state in a solution of (B), or It is dissolved in a solvent together with (B). In the case of a solvent in which (A) and (B) are insoluble, (A) and (B) are in the form of a paste dispersed in the solvent or in a swollen state containing the solvent.

The material of the present invention may further include, if necessary,
By including the cell growth factor (D), the treatment period can be further shortened. Cell growth factor (D)
Are substances having an activity of proliferating cells, for example, fibroblast growth factor, transforming growth factor, epidermal growth factor, hepatocyte growth factor, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor,
Nerve growth factor, stem cell factor, leukemia inhibitory factor, bone morphogenetic factor, heparin-binding epidermal growth factor, neurotrophic factor, connective tissue growth factor, angiopoietin, chondromodulin, tenomodulin, cytokines, interleukins, adrenamodulin and A physiologically active peptide such as a natriuretic peptide or the like is used (for example, “Medical Ueda Tissue Engineering” published by Nagoya University Press (1999) pp. 43-51).

Among them, fibroblast growth factor, transforming growth factor, epidermal growth factor, hepatocyte growth factor, platelet-derived Growth factors, insulin-like growth factors, vascular endothelial cell growth factors and bone morphogens are preferred, more preferably fibroblast growth factors, transforming growth factors, epithelial cell growth factors, hepatocyte growth factors, insulin-like growth factors and bone Forming factors, particularly preferred are fibroblast growth factors, transforming growth factors, insulin-like growth factors and osteogenic factors, most preferably fibroblast growth factors.

When using cell growth factor (D),
The content of (D) is 0.1 to 100 parts by weight of (B).
00001 to 500 parts by weight, more preferably 0.0001 to 50 parts by weight, particularly preferably 0.001 to 500 parts by weight.
It is 1 to 10 parts by weight. That is, in this case, the content (parts by weight) of (D) is based on 100 parts by weight of (B).
It is preferably at least 0.00001, more preferably 0.1.
0001 or more, particularly preferably 0.001 or more,
Moreover, it is preferably 500 or less, more preferably 50 or less, particularly preferably 10 or less. When the content of (D) is within this range, the proliferative properties of the cells are further promoted, and the effect of promoting the healing of tissue defects tends to be more fully exerted.

The material of the present invention may further include, if necessary,
Other components (E) include stabilizers (eg, antioxidants), solubilizers (eg, surfactants), pH adjusters (eg, calcium carbonate and calcium phosphate), and thickeners (eg, , Polyethylene glycol, etc.). When the other component (E) is used, the content of the other component (E) is preferably 0.00001 to 100 parts by weight, more preferably 0.0001 to 50 parts by weight, based on 100 parts by weight of (B). Parts by weight, particularly preferably 0.001 to 10 parts by weight. That is, in this case, the content (parts by weight) of (E) is preferably 0.00001 or more, more preferably 0.0001 or more, and particularly preferably 0.001 or more, based on 100 parts by weight of (B). And 100 or less, more preferably 50 or less, and particularly preferably 10 or less.

The method for producing the material of the present invention is not particularly limited. For example, a bioadsorbable solution may be added to a solution of a cell adhesion active substance (A) and, if necessary, a growth factor (D) and / or another component (E). A method of immersing the substance (B), a method of (A) and, if necessary, a method of immersing (B) in a solution of the growth factor (D) and / or other component (E) and then removing the solvent, (A) and Growth factor (D) and / or
Or a method of mixing a solution of another component (E) with a solution of (B) and, if necessary, a growth factor (D) and / or another component (E), a solution of (A), (B) and A method of mixing a solution of a growth factor (D) and / or another component (E) if necessary and then removing the solvent, a powder of (A),
A method of mixing (B) and optionally a solution of growth factor (D) and / or other components (E), a powder of (A), (B) and optionally growth factor (D) and / or other And then removing the solvent after mixing with the solution of the component (E).

The powder of the cell adhesion active substance (A) is obtained by grinding the solid (A) in a mortar or the like, or spraying the solution (A) in a mist state in a container under reduced pressure, a so-called spray. Obtained by a dry method. The average particle diameter of the powder of the cell adhesion active substance (A) is not particularly limited, but is 0.001 μm.
To 1 cm, more preferably 0.001 to 1 cm.
00 μm. The solution of the cell adhesion active substance (A) or the solution of the bioabsorbable substance (B) can be easily obtained by an ordinary method. For example, (A) or (B) is added to a solvent,
A method of uniformly stirring with a stirrer or the like can be applied.

When the methods (1) and (2) are used, the material in the present invention becomes a gel or a solution containing a solvent.
As the solvent used here, the above-mentioned solvent (C) can be used. When the method of the present invention is used, the material of the present invention is in the form of powder, fiber, cloth, film, sponge or block containing no solvent. In the case of the method of (1), the material of the present invention has the same shape as the shape of the bioabsorbable substance (B), and depending on the shape of (B), powder, fiber, cloth, film, sponge or block State.

When the above method is used, the shape changes depending on the method of desolvation. For example, the solution becomes powdery when spray-dried, and becomes film-like when the solution is spread and desolvated in a plane, and the solution is foamed. After freeze-drying and cross-linking if necessary, it becomes sponge-like. The solvent used here is not particularly limited as long as it dissolves the cell adhesion active substance (A) and / or the bioabsorbable substance (B). In addition to the solvent (C), hydrocarbons, ketones ,
Esters, ethers and halogenated hydrocarbons can be used. As the hydrocarbon, a hydrocarbon having 6 to 7 carbon atoms can be used, and examples thereof include hexane, cyclohexane, heptane, benzene, and toluene.

As the ketone, a ketone having 3 to 6 carbon atoms can be used, and examples thereof include acetone, methyl ethyl ketone and methyl isobutyl ketone. As the ester, an ester having 4 to 6 carbon atoms can be used, and examples thereof include ethyl acetate and butyl acetate. As the ether, an ether having 4 to 6 carbon atoms can be used, and examples thereof include diethyl ether, diethylene glycol, and triethylene glycol. As the halogenated hydrocarbon, a hydrocarbon having a halogen atom and having 1 to 6 carbon atoms can be used, and examples thereof include chloroform, dichloromethane, carbon tetrachloride, and perfluorohexane.

A conventional method can be applied to spray drying. For example, using a mini-spray dryer Model B-191 manufactured by Shibata Scientific Instruments Co., Ltd., an oven temperature of 50 to 150 ° C. and a reduced pressure of 0.degree. It can be performed at 001 Pa to 50 kPa. The solvent can be removed by a usual method, for example, drying in a normal air dryer, a reduced pressure dryer or a freeze dryer under a pressure of -120 to 200 ° C and 0.001 Pa to atmospheric pressure for 1 to 100 hours. You can do that.

For forming a sponge, a usual method can be applied. For example, a solution containing a cell adhesion active substance (A), a bioabsorbable substance (B) and, if necessary, (C) to (E) is prepared by using a polytron. And the like, while stirring the foam using a high-speed stirrer, and bubbling 1.5 to 100 times, -120 to -1
After cooling and freezing in a freezer at 0 ° C. for 10 minutes to 50 hours,
It is obtained by reducing the temperature to 0 ° C to 100 ° C over 5 to 100 hours under reduced pressure of 0.01 Pa to 50 kPa. Further, the crosslinking treatment (thermo-crosslinking and / or crosslinking using a crosslinking agent) to be carried out if necessary is carried out in a normal air dryer or a reduced-pressure dryer in a range from 100 to 100%.
250 ° C, 10 minutes to 1 under 0.001 Pa to atmospheric pressure
This is performed by processing for 00 hours. By crosslinking treatment,
For example, the hydroxyl group and the carboxyl group in (B) or the hydroxyl groups are bonded and crosslinked.

The shape of the material of the present invention may be powder, granule, fibrous, cloth, tubular, film, gel, etc. in that the contact area with body fluid or cells is high and the healing period is short. Sponge-like and block-like are preferred, more preferably gel-like and sponge-like, particularly preferably sponge-like.

The method for containing the cell growth factor (D) is as follows.
There is no particular limitation, and a method of adding (D) alone or impregnating the solution of (D) with a material composed of the cell adhesion active substance (A) and the bioabsorbable substance (B), and desolvating as necessary. Or a method of mixing (D) with the solution of (A) or the solution of (B), mixing these, and removing the solvent as necessary. The method for incorporating the other component (E) is also not particularly limited, and either (E) alone may be added to the material comprising the cell adhesion active substance (A) and the bioabsorbable substance (B), or A method of impregnating the solution and removing the solvent if necessary, a method of mixing (E) with the solution of (A) or the solution of (B), mixing these and removing the solvent as necessary, and the like. No.

The material of the present invention may be subjected to a sterilization treatment if necessary. The sterilization method may be any method that is medically acceptable, and examples include radiation sterilization, ethylene oxide gas sterilization, dry heat sterilization, autoclave sterilization, and dry heat sterilization. The material of the present invention can be used as an implantable tissue regeneration material. Renewable tissues include, for example, skull, muscle, skin tissue,
Bone, cartilage, blood vessels, nerves, tendons, ligaments, follicular tissue, mucosal tissue, periodontal tissue, dentin, bone marrow, retina, serosa, gastrointestinal tract, fat, and other tissues, as well as lung, liver, pancreas, and kidney Organs and the like.

As a specific regeneration method, for example, a material of the present invention was placed in a skull defect portion caused by a skull cut in brain surgery in a volume that is about 1 to 1.5 times the size of the defect portion. The method of regenerating and treating the skull by suturing or the like after the skull or a defect portion of a tissue or an organ due to trauma, the amount of the material of the present invention in a volume of about 1 to 1.5 times the size of the defect portion A method of regenerating and treating the tissue or the organ by suturing or the like after the installation and embedding the tissue or the organ is exemplified.

The material of the present invention can be injected into necessary parts by injection or a catheter or the like in the form of particles or a dispersion dispersed in water as it is, or can be formed into a rod-like or hollow fiber-like molded body by a catheter or the like. It is also possible to install it at the required site. In the case of a dispersion, the concentration of the material of the present invention is preferably 5 to 90% by weight, more preferably 10 to 70% by weight, and particularly preferably 20 to 60% by weight, based on the weight of the dispersion. That is, in this case, the concentration (% by weight) of the material of the present invention is preferably 5 or more, more preferably 10 or more, particularly preferably 20 or more, and preferably 90 or less, based on the weight of the dispersion. It is more preferably at most 70, particularly preferably at most 60. The lot-shaped or hollow fiber-shaped molded body can be molded by an ordinary method, and for example, a method such as extrusion molding or blow molding can be applied.

[0054]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 2 μg of Pronectin F manufactured by Sanyo Kasei Kogyo Co., Ltd. ground into a fine powder (spherical with a particle diameter of about 1 μm) by grinding in a mortar.
And an aqueous acetic acid solution of collagen manufactured by Nitta Gelatin Co., Ltd. (collagen concentration: 0.3% by weight, pH 3.0) 200
mg was added to a 15 ml Spitz tube, 10 mg of chloroform was further added, and the mixture was stirred for 3 minutes at 12,000 rpm using a polytron under ice-water cooling, and the size of the voids was 80 μm on average and a whip having a distribution of 5 to 150 μm. (Flowable containing air bubbles) was obtained.

Next, this was connected to a metal cylindrical tube (with an inner diameter of 18).
2.5 ml) and frozen in a freezer at −80 ° C. for 1 hour.
The temperature was raised to 25 ° C. over 12 hours under reduced pressure of Pa, further dried at 25 ° C. under reduced pressure at 25 ° C. for 60 hours, and further heated in a reduced pressure drier at 140 ° C. under reduced pressure of 0.05 Pa for 12 hours to perform crosslinking treatment. As a result, a material 1 for in-vivo regeneration of a cylindrical sponge-like tissue having a diameter of 18 mm and a thickness (height) of 10 mm was obtained. FIGS. 1 and 2 show micrographs of a cross section in the height direction.

<Tissue regeneration experiment; mouse dermis defect> After shaving the back of the mouse, the epidermis was incised using a scalpel and scissors to remove the entire 15 mm diameter circular dermis layer (about 1 mm thick). Created a defect. Next, the material for in vivo regeneration of a cylindrical sponge-like tissue having a diameter of 18 mm and a thickness (height) of 10 mm obtained in Example 1 was embedded in the dermis defect portion from the incision portion using tweezers, and the epidermis was sutured. 5
One day later, the mouse was anesthetized and killed, and then a tissue specimen was prepared including the surrounding tissue in a columnar shape having a diameter of 30 mm and a thickness of 10 mm centering on the tissue of the implant. Two mice (mouse numbers 1 and 2) with respect to tissue regeneration material 1
Was conducted.

FIG. 3 shows a photograph of a cross section of the tissue specimen (mouse 1) of Example 1. The tissue regeneration state was evaluated according to the following criteria. Table 1 shows the results. ◎: A very good regenerating state in which fibroblasts penetrate the entire sponge and some blood vessels are regenerated. ；: Fibroblasts invaded in more than half of the sponge and in a good regenerating state. Δ: Fibroblast invasion is less than half of the sponge,
The playback state is bad.

Example 2 Pronectin F coating of PGA nonwoven fabric: PGA nonwoven fabric manufactured by Gunze Co., Ltd. (trade name: Neoveil, thickness:
1.3～1.4Mm, Density: 405~410g / m ²⁾
Was cut into 12 × 12 mm ² , and 10 μg of Pronectin F was
/ ML concentration in phosphate buffered saline (PBS) solution. After one hour, take out the PGA nonwoven fabric,
It was left at 5 ° C. for 24 hours. After washing this with PBS twice,
Dry under reduced pressure (10 Pa reduced pressure, 25 ° C., 24 hours)
Furthermore, Pronectin F-coated PGA nonwoven fabric was prepared by sterilizing ethylene oxide-containing gas (ethylene oxide volume concentration: 20%, 40 ° C., 7 hours).

Preparation of freeze-dried gelatin hydrogel: An acidic treated gelatin (isoelectric point: 5.0, molecular weight: 99000) derived from bovine bone manufactured by Nitta Gelatin Co., Ltd. was adjusted to 37 ° C. in distilled water at 5% by weight. And dissolved. 40m of this solution
100 mL of a 25% glutaraldehyde aqueous solution manufactured by Nacalai Tesque, Inc. was added to L, and the mixture was stirred at 37 ° C. The mixture was immediately cast on a balance dish and subjected to a crosslinking reaction at 4 ° C. for 24 hours. The obtained hydrogel is treated with a 0.1 M glycine aqueous solution for 1 hour, washed twice with water, and freeze-dried (10%).
The pressure was reduced to Pa at −80 ° C. for 48 hours, and the mixture was sterilized with ethylene oxide-containing gas (ethylene oxide concentration: 20% by volume, 40 ° C., 7 hours) to prepare a lyophilized gelatin hydrogel.

Preparation of tissue-in-vivo regeneration material 2: A freeze-dried gelatin hydrogel is prepared into a circular shape having a thickness of 1.5 mm and a diameter of 8 mm, and 10 μL of an aqueous solution containing bFGF at a concentration of 1 mg / mL is dropped on the hydrogel. And allowed to stand at 4 ° C. for 12 hours. By sandwiching this bFGF-impregnated gelatin hydrogel between two Pronectin F-coated PGA nonwoven fabrics, a tissue regenerating material 2 in the body was produced.

<Tissue regeneration experiment; mouse dermis defect> The skin on the back of the mouse was incised using a scalpel and scissors, and a pocket-shaped one 12 × 12 mm ² in size was prepared subcutaneously. The tissue regenerating material 2 of the tissue was implanted subcutaneously using tweezers, and the skin was sutured. Seven days later, as in Example 1, two mice (mouse number 3, mouse number 3)
The experiment of 4) was performed to prepare a tissue specimen. A photograph (mouse 3) of a cross section of the tissue specimen of Example 2 is shown in FIG. Also,
The tissue regeneration state was evaluated in the same manner as in Example 1. Table 1 shows the results.

Comparative Example 1 A material 3 for in vivo regeneration of a sponge-like tissue for comparison was prepared in the same manner as in Example 1 except that Pronectin F was not used.
I got Micrographs of the cross section in the height direction are shown in FIGS.
Shown in It can be seen that it has the same structure as the tissue regeneration material 1 of Example 1.

<Tissue regeneration experiment; mouse dermis defect> An experiment was performed on two mice (mouse Nos. 5 and 6) in the same manner as in Example 1 with respect to the tissue regeneration material 3 in the body to prepare a tissue specimen. did. A photograph (mouse 5) of a cross section of the tissue specimen of Example 2 is shown in FIG. The tissue regeneration state was evaluated in the same manner as in Example 1. Table 1 shows the results.

Comparative Example 2 A material 4 for in vivo regeneration of a tissue for comparison was obtained in the same manner as in Example 2 except that Pronectin F was not used.

<Tissue regeneration experiment; mouse dermis defect> An experiment was performed on two mice (mouse Nos. 7 and 8) on the tissue regeneration material 4 in the same manner as in Example 2 to prepare a tissue specimen. did. A photograph (mouse 7) of a cross section of the tissue specimen of Example 2 is shown in FIG. The tissue regeneration state was evaluated in the same manner as in Example 1. Table 1 shows the results.

[0066]

[Table 1]

It can be seen that when the material of the present invention is used, very good tissue regeneration can be obtained with good reproducibility.

[0068]

The use of the tissue regeneration material of the present invention in vivo allows for the healing and regeneration of defective tissue in a very short period of time without any need for extracellular cell culture. In particular, tissues such as muscle, skin tissue, bone, cartilage, blood vessels, nerves, tendons, ligaments, follicular tissue, mucosal tissue, periodontal tissue, dentin, bone marrow, retina, serosa, gastrointestinal tract and fat, lung, liver It has an extremely high effect on the regeneration of organs such as pancreas and kidney.

[0069]

[Brief description of the drawings]

FIG. 1 is a micrograph (magnification: 200 ×) of one section of a tissue regeneration material of the present invention obtained in Example 1 in vivo.

FIG. 2 is a micrograph (magnification: 40) of one section of the tissue regeneration material of the present invention obtained in Example 1 in vivo.

FIG. 3 is a micrograph (magnification: 200 times) of a cross section of a tissue specimen regenerated using the tissue regeneration material 1 of the present invention obtained in Example 1 in vivo.

FIG. 4 is a micrograph (magnification: 58) of a cross section of a tissue specimen regenerated using the tissue regenerating material 2 of the tissue of the present invention obtained in Example 2.

FIG. 5 is a micrograph (magnification: 200 times) of a cross section of a material for in vivo regeneration of a tissue obtained in Comparative Example 1.

FIG. 6 is a micrograph (magnification: 40) of a cross section of a material for in vivo regeneration of tissue obtained in Comparative Example 1.

FIG. 7 is a cross-sectional micrograph (200 × magnification) of a tissue specimen regenerated using the tissue regeneration material 3 for tissue obtained in Comparative Example 1.
It is.

FIG. 8 is a micrograph (magnification: 58 times) of a cross section of a tissue specimen regenerated using the tissue regenerating material 4 obtained in Comparative Example 2;
It is.

[0070]

<Sequence list> <110> Sanyo Chemical Industries, Ltd .; SANYO CHEMICAL INDUSTRIES, LTD. <120> Materials for tissue regeneration in the body <160> 12 <210> 1 <211> 4 <212> PRT <213> Homo sapiens < 400> 1 Arg Glu Asp Val 1 <210> 2 <211> 5 <212> PRT <213> Homo sapiens <400> 2 Tyr Ile Gly Ser Arg 1 5 <210> 3 <211> 5 <212> PRT <213 > Homo sapiens <400> 3 Pro Asp Ser Gly Arg 1 5 <210> 4 <211> 7 <212> PRT <213> Homo sapiens <400> 4 Arg Tyr Val Val Leu Pro Arg 1 5 <210> 5 <211 > 6 <212> PRT <213> Homo sapiens <400> 5 Leu Gly Thr Ile Pro Gly 1 5 <210> 6 <211> 10 <212> PRT <213> Homo sapiens <400> 6 Arg Asn Ile Ala Glu Ile Ile Lys Asp Ile 1 5 10 <210> 7 <211> 5 <212> PRT <213> Homo sapiens <400> 7 Ile Lys Val Ala Val 1 5 <210> 8 <211> 4 <212> PRT <213> Homo sapiens <400> 8 Asp Gly Glu Ala 1 <210> 9 <211> 6 <212> PRT <213> Bombyx mori <400> 9 Gly Ala Gly Ala Gly Ser 1 5 <210> 10 <211> 54 <212 > PRT <213> Artificial Sequence <400> 10 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 1 5 10 15 Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala 20 25 30 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 35 40 45 Gly Ala Gly Ala Gly Ser 50 <210> 11 <211> 4 <212> PRT <213> Homo sapiens <400> 11 Arg Gly Asp Ser 1 <210> 12 <211> 5 <212> PRT <213> Homo sapiens <400> 12 Gly Arg Gly Asp Ser 1 5

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Yuto Kurokawa 1 at 11-11 Hitotsubashi Honcho, Higashiyama-ku, Kyoto City, Kyoto Prefecture Inside (72) Inventor Tatsuya Osumi 11th address, Hitotsubashi Nohonmachi, Higashiyama-ku, Kyoto City, Kyoto Prefecture 1 F-term in Sanyo Chemical Industries Co., Ltd. (reference) 4C081 AB01 AB11 BA12 CA171 CD03 CD04 CD07 CD08 CD09 CD11 CD12 CD15 CF01 CF02 CF03 DA01 DA02 DA03 DA04 DA05 DA11 DA12 DB03

Claims (9)

[Claims] 1. A tissue regeneration material in a body, comprising a cell adhesion active substance (A) and a bioabsorbable substance (B). 2. A polypeptide (A1), which is synthesized by a genetically modified microorganism and has at least one amino acid sequence in one molecule which represents a cell adhesion signal.
The material according to claim 1, which is: 3. The material according to claim 2, wherein the number of minimum amino acid sequences representing a cell adhesion signal in (A1) is 3 to 50. 4. An RGD sequence, an LDV sequence, a REDV sequence (1), a YIGSR sequence (2), a PDSGR sequence (3), and a RYVVLPR, wherein the minimum amino acid sequence representing a cell adhesion signal is represented by an amino acid single letter code. Sequence (4), LGTIPG sequence (5), RNIAEIIKD
I sequence (6), IKVAV sequence (7), LRE sequence, D
4. The material according to claim 2, which is at least one kind of sequence selected from the group consisting of a GEA sequence (8) and a HAV sequence. 5. The method of claim 1, wherein (A1) further comprises GAGAGS (9).
3. The amino acid sequence of claim 2, which has at least two amino acid sequences of
4. The material according to any one of 4 above. 6. The material according to claim 1, wherein (B) is a bioabsorbable substance selected from the group consisting of the bioabsorbable substances shown in (1) to (4). (1) Natural polymers Collagen, gelatin, glycosaminoglycan, hyaluronic acid, chondroitin sulfate, fibrin, alginic acid, chitin, chitosan, fibroin, starch, pectin, pectic acid, and their partially decomposed products, oxides, alkylene oxides Adducts, carboxymethylated products and crosslinked products. (2) Synthetic polymer (co) polymer comprising a monomer selected from the group consisting of lactic acid, glycolic acid and ε-caprolactone as an essential monomer, and the minimum amino acid sequence representing a cell adhesion signal in one molecule Has at least one polypeptide (A
Synthetic polypeptides other than 1). (3) Inorganic substances Tricalcium phosphate, hydroxyapatite and calcium carbonate. (4) Complex comprising natural polymer and synthetic polymer Block and / or graft copolymer obtained by polymerizing lactic acid, glycolic acid and / or ε-caprolactone on a natural polymer. 7. The material according to claim 1, wherein the amount of (A) is 0.0001 to 50 parts by weight per 100 parts by weight of (B). 8. A powdery, granular, fibrous, textile, tubular,
The material according to any one of claims 1 to 7, which is in the form of a film, a gel, a sponge, or a block. 9. The material according to claim 1, further comprising a cell growth factor.