HK1234669A1 - Bone formation promoter - Google Patents

Description

Bone formation promoting material

Technical Field

The present invention relates to a bone formation promoting material. More specifically, the present invention relates to a bone formation promoting material containing a self-assembling peptide and a bone fragment.

Background

The spine not only has a function of supporting the body weight but also has a function of protecting the spinal cord, which is the central nerve. In the field of orthopedic surgery, particularly, the spine, diseases that affect the nerves such as the spinal cord (for example, cervical spondylotic myelopathy) and fractures of the spine become a great problem. The spinal cord is not restricted to a method of cutting the bone that presses the spinal cord. In general, a spine which is unstable due to resection is implanted with a bone collected by itself or fixed with a metal such as a metal bolt or a plate.

In addition, the basic methods of fracture treatment are reduction and fixation, and a method of fixing using metal to allow bone healing is generally used. However, in the case of a fracture of the limbs, a long-term healing period of 2 to 3 months is required, and a long-term healing period of half a year to 1 year or more is required for a fracture of the spine. Among them, in the case of an elderly person, since the bone itself becomes weak, there is a possibility that the bone is fractured again before bone healing, or that a metal used for fixation is deviated. In addition, femoral neck fractures or vertebral fractures sometimes require a quiet bed, and therefore, disuse syndrome may also occur and require care.

In recent years, various surgical methods and fixing materials have been developed, and the cure rate of bone fracture has been improved. However, sequelae such as delayed healing (delayed bone healing) or pseudoarthrosis (cessation of bone healing) still occur. In addition, in spinal surgery, more effective implantation or surgical techniques are being developed in spinal fixation for spinal reconstruction. However, when used for elderly people with reduced bone regeneration function, there is a possibility that bone healing may not be properly performed.

In recent years, as a method for promoting bone regeneration, a method of transplanting cultured cells such as osteoblasts into an affected part, a method of placing a cell scaffold on an affected part, or the like has been attempted. As the cell scaffold, for example, an extracellular matrix typified by gelatin and collagen can be used, but gelatin and collagen have a disadvantage that their use is limited depending on animals or the like as a material supply source. On the other hand, it has been reported that when a completely synthesized self-assembling peptide is administered alone or in combination with a bone differentiation-inducing factor, bone regeneration is promoted (patent document 1, patent document 2, patent document 3, and non-patent document 1). However, it is difficult to ensure sufficient strength under neutral conditions close to the in vivo environment in a bone formation promoting material containing the self-assembling peptide which has been reported so far to promote bone regeneration.

Documents of the prior art

Patent document

Patent document 1: japanese laid-open patent publication No. 2012-82180

Patent document 2: japanese Kokai publication No. 2010-504972

Patent document 3: international publication No. 2007/000979

Non-patent document

Non-patent document 1: cell Transplantation, Vol.15, p903-910, 2006

Disclosure of Invention

Problems to be solved by the invention

The purpose of the present invention is to provide a bone formation promoting material which is highly safe in living body and can promote bone formation.

Means for solving the problems

The bone formation promoting material of the present invention contains self-assembled peptides and bone fragments that can form a beta-sheet structure in an aqueous solution at neutral pH. The sum of the charges of the amino acid residues constituting the self-assembling peptide at pH7.0 is not 0.

In one embodiment, the self-assembling peptide is a self-assembling peptide comprising the following amino acid sequence.

Amino acid sequence: a is₁b₁c₁b₂a₂b₃db₄a₃b₅c₂b₆a₄

(in the amino acid sequence, a₁～a₄Is a basic amino acid residue; b₁～b₆Are uncharged polar amino acid residues and/or hydrophobic amino acid residues, and at least 5 of which are hydrophobic amino acid residues; c. C₁And c₂Is an acidic amino acid residue; d is a hydrophobic amino acid residue. )

In one embodiment, the bone formation promoting material further contains blood and/or a blood-derived component.

In one embodiment, the bone formation promoting material further comprises a bone formation factor.

In one embodiment, the bone formation promoting material is used in combination with a holding tool for a bone formation promoting material.

In other aspects of the present invention, a holding tool for a bone formation promoting material is provided. The bone formation promoting material holding instrument of the present invention is used together with the bone formation promoting material.

In one embodiment, the holder for a bone formation promoting material is made of a carbon-based material, an engineering resin, or a super engineering resin.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention provides a bone formation promoting material which is highly safe in living body and can promote bone formation. The bone formation promoting material of the present invention contains self-assembled peptides and bone fragments that can form a beta-sheet structure in an aqueous solution at neutral pH. This forms a lot of starting points for bone formation, and can promote bone formation well. Therefore, bone formation can be improved even in an elderly person whose strength of bone itself and bone regeneration ability are reduced. In addition, the bone formation promoting material of the present invention can induce osteogenesis without using cells directly involved in bone formation or the like. Further, the self-assembling peptide used in the present invention forms a β sheet structure in an aqueous solution at neutral pH, and the total charge of the amino acid residues constituting the self-assembling peptide at pH7.0 is not 0. Therefore, the bone formation promoting material of the present invention can exhibit sufficient strength at a neutral pH close to the living environment. Further, by using an appropriate holder such as the holder for a bone formation promoting material of the present invention, the bone formation promoting material can be fixed at a desired position.

Drawings

FIG. 1A is a photograph of MC3T3-E1 cells cultured for 0 day, a photograph of MC3T3-E1 cells cultured for 1 day, and a photograph showing ALP staining.

FIG. 1B is a photograph of MC3T3-E1 cells cultured for 7 days and a photograph showing ALP staining.

FIG. 1C is a photograph of MC3T3-E1 cells cultured for 14 days and a photograph showing ALP staining.

FIG. 1D is a photograph of MC3T3-E1 cells cultured for 28 days and a photograph showing ALP staining.

FIG. 2 is a graph showing the expression levels of bone formation markers in cells after 1 day, 7 days, 14 days, and 28 days of culture.

FIG. 3 is a photograph of the bone formation promoting materials of examples 1 to 3 and a photograph of the bone formation promoting material of comparative example 1 after 14 days and 28 days of culture and an ALP-stained photograph.

Fig. 4 is a photograph of an external fixator used for modeling a femoral defect.

Fig. 5 is a photograph of a cage (cage) used for modeling a femoral defect.

Fig. 6 is a photograph of the femur of a mouse with external fixation.

Fig. 7 is an X-ray image of the femur of a mouse after 56 days of external fixation.

Fig. 8 is a CT image of mouse femurs after 56 days of external fixation.

Fig. 9(a) is a photograph of a gel before adding a bone fragment (born chip) of example 5, fig. 9(b) is a photograph of a gel before adding a bone fragment of example 6, and fig. 9(c) is a photograph of a gel before adding a bone fragment of comparative example 2.

Fig. 10(a) is a photograph of a gel (bone formation promoting material) after addition of a bone fragment of example 5, fig. 10(b) is a photograph of a gel (bone formation promoting material) after addition of a bone fragment of example 6, and fig. 10(c) is a photograph of a gel (bone formation promoting material) after addition of a bone fragment of comparative example 2.

Detailed Description

[ A. bone formation promoting Material ]

The bone formation promoting material of the present invention contains self-assembled peptides and bone fragments that can form a beta-sheet structure in an aqueous solution at neutral pH. The sum of the charges of the amino acid residues constituting the self-assembling peptide at pH7.0 is not 0. In one embodiment, the bone formation promoting material of the present invention further comprises blood and/or a blood-derived component. In another embodiment, the bone formation promoting material of the present invention further comprises a bone formation factor.

The bone formation promoting material of the present invention has a storage elastic modulus (G') at 37 ℃ of, for example, 1Pa to 5000Pa, preferably 1Pa to 4000Pa, and more preferably 1Pa to 3000Pa, as measured by a rotary rheometer for dynamic viscoelasticity. When the storage elastic modulus is within this range, the bone formation promoting material can be used in combination with a holder for a bone formation promoting material of the present invention described later, and thus bone formation can be favorably promoted. The storage modulus of elasticity as used herein means a value obtained when the number of angular vibrations measured by measuring a change in frequency is 1 rad/sec.

[ A-1. self-assembling peptide ]

As the self-assembling peptide, any suitable peptide that spontaneously aggregates by the interaction of peptide molecules with each other in an aqueous solution and can form a gel may be used. The self-assembling peptides used in the present invention can form a beta sheet structure at neutral pH. More specifically, it is preferable to use a peptide which spontaneously aggregates to form a fibrous molecular aggregate by the interaction of peptide molecules in an aqueous solution and can form a gel by the development of a three-dimensional network structure by the interaction of the molecular aggregate. Examples of interactions between peptide molecules include: electrostatic interactions such as hydrogen bonding, ionic interactions, van der waals forces, and hydrophobic interactions. In the present specification, the term "neutral pH (neutral region)" means: a pH of 5.0 to 8.0, preferably 5.5 to 7.5, more preferably 6.0 to 7.0, and still more preferably 7.0.

The amino acids constituting the self-assembling peptide may be L-amino acids or D-amino acids. L-amino acids are preferred. The amino acid may be a natural amino acid or an unnatural amino acid. Natural amino acids are preferable because they can be obtained at low cost and peptide synthesis is easy.

The sum of the charges of the amino acid residues constituting the self-assembling peptide at pH7.0 is not 0. The sum of the charges is preferably-3 to-1 or +1 to +3, more preferably-3, -2, +2 or + 3. This is because, in this case, the positive charge and the negative charge derived from the side chain of the amino acid residue contained in the self-assembling peptide do not cancel each other in the neutral region, and therefore, the balance between the electrostatic attraction force and the repulsive force suitable for gel formation is maintained, and as a result, a transparent and stable gel can be formed in the neutral region.

The charge of the self-assembling peptide at each pH can be carried out by an available program on the website of PROTEIN calcutter v3.4 (http:// protcalc. source for. net /).

Specific examples of the self-assembling peptide which can be preferably used in the present invention include peptides having an amino acid sequence represented by the following formula (I).

a₁b₁c₁b₂a₂b₃db₄a₃b₅c₂b₆a₄(I)

(in the amino acid sequence, a₁～a₄Is a basic amino acid residue; b₁～b₆Are uncharged polar amino acid residues and/or hydrophobic amino acid residues, and at least 5 of which are hydrophobic amino acid residues; c. C₁And c₂Is an acidic amino acid residue; d is a hydrophobic amino acid residue. )

In the above amino acid sequence, a₁～a₄Is a basic amino acid residue. The basic amino acid is preferably arginine, lysine or histidine, more preferably arginine or lysine. This is because these amino acids are strongly basic. a is₁～a₄The amino acid residues may be the same or different.

In the above amino acid sequence, b₁～b₆Are uncharged polar amino acid residues and/or hydrophobic amino acid residues, at least 5 of which are hydrophobic amino acid residues. The hydrophobic amino acid is preferably alanine, leucine, isoleucine, valine, methionine, phenylalanine, tryptophan, glycine or proline. The uncharged polar amino acid is preferably tyrosine, serine, threonine, asparagine, glutamic acid or cysteine. This is because these amino acids are readily available.

Preferably b₃And b₄Each independently is any suitable hydrophobic amino acid residue, more preferably a leucine residue, an alanine residue, a valine residue, or an isoleucine residue, and particularly preferably a leucine residue or an alanine residue.

Preferably b₁～b₆All are hydrophobic amino acid residues. This is because of self-organizationThe peptide is well formed into β lamellar structures which can self-assemble, more preferably b₁～b₆Each independently represents a leucine residue, an alanine residue, a valine residue, or an isoleucine residue, and more preferably a leucine residue or an alanine residue. In a preferred embodiment, b₁～b₆At least 4 of them are leucine residues, more preferably at least 5 of them are leucine residues, and still more preferably all leucine residues.

In the above amino acid sequence, c₁And c₂Is an acidic amino acid residue. The acidic amino acid is preferably aspartic acid or glycine. This is because these amino acids are readily available. c. C₁And c₂The amino acid residues may be the same or different.

In the above amino acid sequence, d is a hydrophobic amino acid residue. d is preferably an alanine residue, a valine residue, a leucine residue or an isoleucine residue.

In a preferred embodiment, b₃、d、b₄2 of the consecutive 3 amino acid residues of (a) are leucine residues, and the remainder are alanine residues. In this case, b₃、d、b₄May be an alanine residue. In another preferred embodiment, b is₃、d、b₄The consecutive 3 amino acid residues of (a) are all leucine residues.

Preferred specific examples of the amino acid sequence of formula (I) are shown below.

n-RLDLRLALRLDLR-c (SEQ ID NO. 1)

n-RLDLRLLLRLDLR-c (SEQ ID NO. 2)

n-RADLRLALRLDLR-c (SEQ ID NO. 3)

n-RLDLRLALRLDAR-c (SEQ ID NO. 4)

n-RADLRLLLRLDLR-c (SEQ ID NO. 5)

n-RADLRLLLRLDAR-c (SEQ ID NO. 6)

n-RLDLRALLRLDLR-c (SEQ ID NO. 7)

n-RLDLRLLARLDLR-c (SEQ ID NO. 8)

As another self-assembling peptide which can be preferably used in the present invention, there is mentioned a peptide described in WO2007/000979, that is, a self-assembling peptide having a polar amino acid residue and a nonpolar amino acid residue (hydrophobic amino acid residue), wherein the polar amino acid residue contains an acidic amino acid residue and a basic amino acid residue, and the sum of the charge of the acidic amino acid residue and the charge of the basic amino acid residue in a neutral region is a number other than 0, and a β -sheet structure in which the nonpolar amino acid residue is arranged on only one side can be formed when self-assembling is performed in an aqueous solution.

Among the above self-assembling peptides, the polar amino acid is preferably a peptide containing an acidic amino acid residue, a basic amino acid residue and an uncharged polar amino acid. Preferred specific examples of the self-assembling peptide are shown below.

n-RASARADARASARADA-c (SEQ ID NO. 9)

n-RANARADARANARADA-c (SEQ ID NO. 10)

n-RAAARADARAAARADA-c (SEQ ID NO. 11)

n-RASARADARADARASA-c (SEQ ID NO. 12)

n-RADARASARASARADA-c (SEQ ID NO. 13)

n-RASARASARASARADA-c (SEQ ID NO. 14)

n-RASARADARASA-c (SEQ ID NO. 15)

n-KASAKAEAKASAKAEA-c (SEQ ID NO. 16)

n-SAEAKAEASAEAKAEA-c (SEQ ID NO. 17)

n-KLSLKLDLKLSL-c (SEQ ID NO. 18)

n-KLALKLDLKLAL-c (SEQ ID NO. 19)

The self-assembling peptide can be produced by any suitable production method. Examples thereof include: chemical synthesis methods such as Fmoc method and the like, liquid phase method and the like, and molecular biology methods such as gene recombination expression and the like.

The self-assembling peptide may be modified as appropriate according to the purpose. The site to be modified is not particularly limited, and examples thereof include: the N-terminal amino group, the C-terminal carboxyl group, or both of the self-assembling peptides.

As the modification, any suitable modification may be selected within the range where the modified peptide has self-assembly ability. Examples thereof include: introduction of a protecting group such as acetylation of an N-terminal amino group and amidation of a C-terminal carboxyl group; introduction of functional groups such as alkylation, esterification, or halogenation; hydrogenation; introduction of a sugar compound such as monosaccharide, disaccharide, oligosaccharide or polysaccharide; introduction of lipid compounds such as fatty acids, phospholipids, and glycolipids; introducing amino acid or protein; introducing DNA; introduction of other physiologically active compounds and the like. The modification may be performed in only 1 kind, or may be performed in 2 or more kinds in combination. For example, the N-terminus of an additional peptide having a desired amino acid introduced into the C-terminus of the self-assembling peptide may be acetylated, and the C-terminus may be amidated.

In the case of introducing an amino acid or a protein, the amount of the introduced amino acid is preferably 1 to 180, more preferably 1 to 50, still more preferably 1 to 30, particularly preferably 1 to 10, and most preferably 1 to 5. When the number of amino acid residues introduced exceeds 180, the self-assembly ability may be impaired.

The bone formation promoter of the present invention may contain only 1 kind of self-assembling peptide, or may contain 2 or more kinds of self-assembling peptides.

The concentration of the self-assembling peptide in the bone formation promoting material of the present invention may be appropriately set depending on the composition, the use, and the like. The concentration of the self-assembling peptide is preferably 0.1 to 5.0 wt%, more preferably 0.1 to 3.0 wt%, and still more preferably 0.1 to 2.0 wt%. When the concentration is within the above range, the effects of the present invention can be obtained favorably.

[ A-2. bone chips ]

The bone fragments used in the present invention may be bones of patients themselves treated with the bone formation promoting material of the present invention, or may be bones of other persons. The bone may be bone collected from a site to which the bone formation promoting material is applied, or bone collected from another site. As the bone used as the bone fragment, for example, a bone fragment cut for removing pressure or the like can be used.

The bone fragments may be used as they are, or may be used after any suitable pretreatment. Examples of the pretreatment include washing and drying by perfusion with Phosphate Buffered Saline (PBS).

The size of the bone fragments is not particularly limited, and may be any suitable size depending on the site to which the bone formation promoting material is applied. For example, the bone fragments may be used as they are, or the bone fragments may be prepared into slices having a desired thickness, or may be processed into any suitable size (for example, powder) and used. In the case of using the bone fragments as they are, the size of the bone fragments is, for example, 0.5mm to 5.0mm, preferably 1.0mm to 4.0 mm. When processed into any suitable size (for example, powder) and used, the size of the bone fragments is, for example, 0.01 to 0.5mm, preferably 0.02 to 0.4 mm. The size of the bone fragments is preferably 0.03mm to 0.3mm, more preferably 0.04mm to 0.2 mm. When the size of the bone fragments is within the above range, bone formation can be promoted more favorably. In the present specification, the size of a bone fragment means the size of the largest-sized portion in one bone fragment.

The bone fragments may be processed to a desired size by any suitable means. Examples thereof include a pulverizer and the like.

The bone fragments in the bone formation promoter of the present invention are contained in an amount of preferably 1 to 100 parts by weight, more preferably 1 to 90 parts by weight, based on 100 parts by weight of the self-assembling peptide gel. When the content of the bone fragments is within the above range, bone formation can be promoted more favorably. In the present specification, the reference self-assembling peptide gel refers to a gel composed of only self-assembling peptides and water.

[ A-3. blood and blood-derived component ]

The bone formation promoting material of the present invention preferably further contains blood and/or a blood-derived component. By further containing blood and/or a component derived from blood, an effect of promoting the induction of cartilage formation can be obtained. Examples of the blood-derived component include: red blood cells, white blood cells, platelets, plasma, and the like. These blood-derived components may be used alone, or 2 or more kinds thereof may be used in combination.

The blood and/or blood-derived components can be collected from the patient or from another person. In addition, blood preparations such as whole blood preparations, red blood cell preparations, plasma preparations, and platelet preparations can be used.

The concentration of blood and/or blood-derived components in the bone formation-promoting material of the present invention is preferably 0.001ppm to 1000ppm, more preferably 0.01ppm to 1000 ppm. When the concentration of blood and/or a blood-derived component is within the above range, bone formation can be more favorably promoted. [ A-4. bone Forming factor ]

The Bone formation promoting material of the present invention preferably further contains a Bone Morphogenetic Protein (BMP). The bone formation promoting material of the present invention can further promote bone formation by further containing a bone formation factor. Specific examples of the bone-forming factor include: BMP2/4 family BMPs such as BMP2 and BMP 4; OP-1-based BMPs such as BMP5, BMP6, BMP7, BMP8a and BMP8 b; BMP 9-based BMPs such as BMP9 and BMP 10; BMP of GDF5 series such as GDF5, GDF6, GDF7, etc. The bone-forming factor is preferably BMP-2 in that it can promote bone formation well. In addition, the bone formation factor may be appropriately modified within a range having a bone formation promoting effect equivalent to that of the bone formation factor. In addition, human recombinant bone morphogenetic factor (rhBMP) can be used as the bone morphogenetic factor. The bone formation factors may be used alone, or 2 or more kinds thereof may be used in combination. Since these human BMPs have been cloned, they can be obtained by genetic engineering methods based on their base sequences.

The concentration of the bone formation factor in the bone formation promoting material of the present invention is usually 0.001ppm to 1000ppm, preferably 0.01ppm to 1000 ppm.

When the concentration is within this range, the action of the physiologically active substance is favorably exerted, and bone formation is promoted.

[ A-5. other physiologically active substances ]

The bone formation promoting material of the present invention may further contain a physiologically active substance other than the above-mentioned bone formation factor. Examples of the physiologically active substance that can be contained in the bone formation promoting material of the present invention include: differentiation control factors (e.g., TGF- β) that induce or promote differentiation of bone or cartilage; a growth hormone; cell function control factors such as EGF and FGF; immune or inflammation-related factors such as interferons and interleukins; and the like.

The concentration of the physiologically active substance in the bone formation promoting material of the present invention is usually 0.001ppm to 1000ppm, preferably 0.01ppm to 1000 ppm. Within this concentration range, the physiologically active substance can act favorably to promote bone formation.

[ A-6. other additives ]

The bone formation promoting material of the present invention may further contain any appropriate additive as required. Specific examples of the additives include: a pH adjusting agent; a buffering agent; a tonicity agent; salts; amino acids; vitamins; alcohols; a protein; drugs, and the like. These additives may be used alone, or 2 or more of them may be used in combination.

Examples of the pH adjuster include: hydrochloric acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, and the like.

Examples of the buffer include: phosphates such as phosphoric acid, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate; boric acid, borax, sodium borate, potassium borate, and the like; citrate such as sodium citrate and disodium citrate; acetate salts such as sodium acetate and potassium acetate, Tris and HEPES, and the like.

Examples of tonicity agents include: chlorides such as sodium chloride, potassium chloride, calcium chloride, and magnesium chloride; monosaccharides such as glucose, fructose, and galactose; disaccharides such as sucrose, trehalose, maltose, and lactose; sugar alcohols such as mannitol and sorbitol; and the like.

As the salt, any suitable salt other than the additives exemplified above can be used. Examples thereof include sodium sulfate and magnesium sulfate.

The amount of the additive may be set to any appropriate value according to the purpose thereof.

[ B. production method ]

The bone formation promoting material of the present invention may be prepared by any suitable method. For example, the self-assembling peptide gel is prepared by mixing the self-assembling peptide, water and other optional ingredients and then leaving to stand, and the gel is mixed with bone chips. The self-assembling peptide used in the present invention spontaneously aggregates in a solution by the interaction of peptide molecules to form a fibrous molecular assembly, and further, is allowed to stand still, whereby a three-dimensional network structure develops by the interaction between the molecular assemblies to take the form of a gel. The standing time and the standing temperature can be appropriately set according to the administration target, the concentration and the type of the self-assembling peptide, and the like. As the water, purified water such as ion-exchanged water and distilled water can be preferably used.

When a small-sized bone fragment (for example, a powdery bone fragment) is used as the bone fragment, it is preferable to mix the self-assembling peptide gel with the bone fragment in order to uniformly disperse the bone fragment. In the case of using a large-sized bone fragment (for example, a bone fragment of about 3 mm) as the bone fragment, the self-assembling peptide gel may be mixed with the bone fragment, or the gel may not be mixed with the bone fragment (the bone fragment may be added only to the gel). The mixing method is not particularly limited as long as it is mixed by any appropriate means.

The method for preparing a bone formation promoting material of the present invention may further comprise: refining by filtration and the like; sterilizing by high pressure steam, radioactive rays, dry heat, etc.; dispensing into a packaging container; and the like.

[ C. holding device for bone formation promoting Material ]

In one embodiment, the bone formation promoting material of the present invention is used together with a holding tool for a bone formation promoting material. The bone formation promoting material has sufficient strength at a neutral pH close to the in vivo environment. By fixing the bone formation-promoting material with the holding instrument of the present invention, the bone formation-promoting material can be held at the administration site. This can promote bone formation in the target administration site, for example, can promote bone healing.

The bone formation promoting material holding device is preferably a net or a device having a mesh-like structure. In such an embodiment, the holding tool of the present invention can hold the promotion of bone formation without applying an excessive load to the administration site. Therefore, for example, even in an elderly person whose strength of the bone itself is reduced, the bone can be suitably used without placing a burden on the bone. In addition, in the case of fracture, it is difficult to fix the fracture at a site having a complicated shape. However, since the retainer for a bone formation promoting material of the present invention can use a net, it can follow a site having a complicated shape and can hold a bone formation promoting material well. Examples of the device having a mesh-like structure include a stent.

The holder for a bone formation promoting material of the present invention is preferably made of a carbon-based material in view of excellent biosafety. As the carbon-based material, any suitable material can be used. Examples of the carbon-based material include carbon-based fibers and carbon nanotubes. The carbon-based material is preferably a carbon-based fiber or a carbon nanotube.

In addition, any suitable plastic graft product can be used as the holder for the bone formation promoting material. Specifically, a cage (cage), a screw (screw), a rod (rod), and the like can be mentioned.

The orthopedic implant article can be constructed of any suitable material. Examples thereof include: metals such as pure titanium, titanium alloy, titanium-nickel alloy, cobalt-chromium, and polycarbonate resin; a polyacetal resin; polyester resins such as polyethylene terephthalate, polybutylene terephthalate, and polycyclohexylidene dimethyl terephthalate; a polyphenylene ether resin; polyphenylene ether; polyamide resins such as nylon 6, nylon 66, and aromatic polyamide; syndiotactic polystyrene; engineering resins such as ultrahigh molecular weight polyethylene, and super engineering resins such as polyphenylene sulfide resins, polysulfone resins, polyethersulfone resins, polyetheretherketone resins, polyarylate resins, liquid crystal polymers, aromatic polyester resins, polyimide resins, polyamideimide resins, polyetherimide resins, and aromatic polyamide resins. From the viewpoint of high heat resistance, mechanical strength, chemical resistance and excellent wear resistance, it is preferable to use a plastic graft product made of an engineering resin or a super engineering resin. The engineering resin or super engineering resin may contain a reinforcing material such as carbon fiber.

[ method of Using bone formation promoting Material ]

The bone formation promoting material of the present invention is administered in the form of a gel in one embodiment, and it is preferable to use a small-sized bone fragment (for example, a powdery bone fragment). Therefore, the composition can be easily administered with a syringe or the like, and can be filled in a complicated or narrow administration target site (for example, a gap in a bone fracture).

In another embodiment, the bone formation promoting material of the present invention is used by gelling the bone formation promoting material and then administering (injecting) the gelled material to a site to be administered. By gelling the bone formation-promoting material, it is possible to maintain a state in which the bone fragments are well dispersed in the bone formation-promoting material. Therefore, the bone formation promoting effect of the bone formation promoting material of the present invention can be exhibited well.

The injection into the site to be administered with the bone formation promoting material may be carried out by any appropriate means such as a syringe, a tube, or a pipette.

The self-assembling peptide contained in the bone formation promoting material of the present invention is excellent in biodegradability. The bone fragments contained in the bone formation promoting material of the present invention promote bone formation and contribute to the formation of new bone. In addition, the bone fragments contained in the bone formation promoter of the present invention remain at the site to be administered even after the self-assembling peptide is decomposed, and can become a part of a bone to be healed.

In another embodiment, the bone formation promoting material of the present invention is used together with the above-described holder for a bone formation promoting material. For example, after a gel-like bone formation promoting material is administered to a subject site, the subject site and the bone formation promoting material are covered with a mesh-like holding tool for a bone formation promoting material, whereby the bone formation promoting material can be held. Further, the bone formation promoting material may be filled in a holding tool (e.g., a cage) for a bone formation promoting material, and then the holding tool may be attached to the administration site. In addition, a bone formation promoting material in the form of, for example, a gel may be administered to a target site in a state where a holder for a bone formation promoting material (e.g., a stent) is applied to the target site in advance. In this embodiment, the bone formation promoting material and the holding tool may be integrated. However, since the self-assembling peptide contained in the bone formation promoting material of the present invention is excellent in biodegradability, it is absorbed and decomposed with time, and the bone fragments can become a part of a curable bone. Therefore, after completion of bone formation, only the holding tool for a bone formation promoting material may be removed as necessary.

The bone formation promoting material of the present invention may be used in combination with any appropriate fixation method as required. For example, since the spine is a portion to which a large load is constantly applied, more stable fixation is required. Therefore, for example, when the bone formation-promoting material of the present invention is applied to a vertebral fracture, the bone formation is promoted in a stably fixed state by using the bone formation-promoting material of the present invention together with a plate or a screw which is a conventional fixing method, and thus the bone healing can be promoted in a more appropriate state.

The bone formation promoting material of the present invention is useful for, for example, repairing a defect site in a bone tissue, a periodontal tissue, or the like. In addition, the bone formation promoting material of the present invention can promote bone formation while reducing the load on the bone. Therefore, the present invention can be suitably applied to elderly people who are concerned about a decrease in bone strength or a decrease in bone regeneration function.

Examples

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.

[ test example 1: cell culture test

A peptide Gel of self-assembled peptide Gel 1 (product name: Panaceta Gel SPG-178-208 (containing peptide (SPG-178) of SEQ ID NO. 1 with N-terminal acetylated and C-terminal amidated (SPG-178) of sequence No. 1 (the sum of charges in the neutral region (pH7.0) constituting the amino acid residues of the self-assembled peptide: 0.8 w/w%) was allowed to stand at 4 ℃ for 1.5 hours, then 500. mu.l of a medium (medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin (PenStrep) solution added to MEM α) was added, the medium was left to stand at 37 ℃ for 1 hour, replaced with a 24-well plate, MC3T 3-E32 cells were seeded at 50,000 cells/well on the 24-well plate added, the medium was cultured at room temperature for 24 hours, and then replaced with an induction medium (further added with 1% ascorbic acid, 0.2% ascorbic acid, and a comparative sample C36-36% hydrocortisone prepared by culture at room temperature, and a comparative method of a medium (Dmax, a mixture of Mitsumadaga) was added to culture medium, and cultured at room temperature^TM”，Ac-RADARADARADARADA-CONH₂Gel with a peptide concentration of 1 wt%) was performed in the same manner as in the self-assembling peptide gel 1 except that the gel was used. In addition, the same is true without adding self-assembling peptide gelCell-only culture was performed. ALP staining and RNA extraction of cells were performed 1 day, 7 days, 14 days, and 28 days after the start of culture. The RNA expression level was determined by RT-PCR. The alkaline phosphatase (ALP) staining was performed by the following method.

(ALP staining)

Staining was performed using Histofine SAB-PO (R) kit (Nichirei Biosciences). The medium was aspirated from each well and washed 3 times with Phosphate Buffered Saline (PBS). Subsequently, the solution was fixed with 4% Paraformaldehyde (PFA), allowed to stand for 10 minutes, and then washed 3 times with PBS. Thereafter, the mixture was permeabilized in a 0.2% Triton X (registered trademark) -PBS solution, left to stand for 10 minutes, and washed 3 times with PBS. After addition of blocking reagent I (3% hydrogen peroxide plus methanol), the mixture was left to stand for 10 to 15 minutes and washed 3 times with PBS. Thereafter, blocking reagent II (10% goat normal serum) was added, followed by standing for 10 minutes, adding a primary antibody (ALP antibody: abcam Anti-alkali protein phospholipid, Tissue Non-Specific [ EPR4477] antibody ab 108337) and standing for 2 hours, followed by washing with PBS 3 times. Subsequently, a secondary antibody (biotin-labeled anti-rabbit IgG antibody) was added and left to stand for 10 minutes, and washed 3 times with PBS. Subsequently, an enzyme reagent (peroxidase-labeled streptavidin) was added and left to stand for 5 minutes, followed by washing 3 times with PBS. Next, 2 drops (about 40. mu.l) of a substrate solution (DAB substrate kit: 2 drops (about 40. mu.l) of a chromogenic substrate (reagent A) and 2 drops (about 40. mu.l) of a substrate buffer (reagent B) were added to 1ml of purified water, and mixed so as to be non-foaming, and 1 drop (about 40. mu.l) of a chromogenic reagent (reagent C) was added to a solution mixed so as to be non-foaming, and the mixture was left to stand for 5 to 20 minutes and washed 3 times with purified water. Subsequently, counterstaining (hematoxylin) was performed, and the sample was left to stand for 2 minutes, developed with water, left to stand for 10 minutes, and observed with a microscope.

< results >

The microphotographs of the cells after 0 day, 1 day, 7 days, 14 days, and 28 days of culture and the photographs stained with ALP are shown in FIGS. 1A to 1D. By ALP staining, ALP as a bone formation marker was stained. When the self-assembled peptide gel 1 was used, it was considered that osteoblasts stained with ALP aggregated and bone formation was sufficiently induced (fig. 1). On the other hand, when only cells are cultured, the cells are not differentiated, and thus staining with ALP is rarely expressed. Even when the self-assembled peptide gel C1 was used, the range of staining with ALP was confirmed (fig. 1).

FIG. 2 is a graph showing the expression levels of bone formation markers (ALP, BMP-2, Bone Sialoprotein (BSP), Osteocalcin (Osteocalcin), Osterix (zinc finger transcription factor), Osteopontin (OSP)) in cells after 1 day, 7 days, 14 days, and 28 days of culture. On day 28 of culture (bone formation maturation period), the expression level was significantly increased in the case of using the self-assembled peptide gel 1 (SPG in fig. 2) compared to the case of culturing using the self-assembled peptide gel C1 (PM in fig. 2) for ALP, BMP-2, OPN, BSP, and Osterix. ALP is generally a marker for an increase in the initial expression level during bone formation. Since ALP is expressed in high amounts as in other bone formation markers, it is considered that each bone formation marker can be continuously induced by using the self-assembling peptide gel 1.

[ example 1]

The femur of a mouse was pulverized by a pulverizer to obtain bone fragments (1mm to 2 mm). The obtained bone fragments were allowed to stand with self-assembling peptide Gel 2 (trade name: Panaceta Gel SPG-178-204, peptide concentration: 0.4 w/w%, manufactured by Menicon) to obtain a Gel-like bone formation promoting material 1. The composition of self-assembling peptide gel 2 is shown in table 1. The obtained bone formation promoting material was cultured using the medium MEM α, 10 m% FBS and 1% Anti-Anti. Fig. 3 shows photographs of the bone formation promoting material after 14 days and 28 days of culture and photographs of the bone formation promoting material stained with ALP.

[ Table 1]

[ example 2]

A bone formation promoting material 2 was obtained in the same manner as in example 1, except that the self-assembling peptide gel 1 was used instead of the self-assembling peptide gel 2. The obtained bone formation promoting material 2 was cultured in the same manner as in example 1. Fig. 3 shows photographs of the bone formation promoting material after 14 days and 28 days of culture and photographs of the bone formation promoting material stained with ALP.

[ example 3]

A bone formation promoting material 3 was obtained in the same manner as in example 1, except that the self-assembling peptide gel 3 was used instead of the self-assembling peptide gel 2. The composition of self-assembling peptide gel 3 is shown in table 1. The obtained bone formation promoting material 3 was cultured in the same manner as in example 1. Fig. 3 shows photographs of the bone formation promoting material after 14 days and 28 days of culture and photographs of the bone formation promoting material stained with ALP.

Comparative example 1

Except that self-assembled peptide gel C1 (product name "Puramatrix" manufactured by 3D matrix Co., Ltd.) was used^TM”，Ac-RADARADARADARADA-CONH₂Gel having a peptide concentration of 1 wt%) was prepared in the same manner as in example 1, except that the self-assembling peptide gel 2 was replaced, and a bone formation promoting material C1 was obtained. The composition of self-assembling peptide gel C1 is shown in table 1. The culture was carried out in the same manner as in example 1, except that the obtained bone formation promoting material C1 was used. Fig. 3 shows photographs of the bone formation promoting material after 14 days and 28 days of culture and photographs of the bone formation promoting material stained with ALP.

(results)

Osteoblasts stained with ALP were confirmed in the bone formation promoting materials 1 to 3. On the other hand, in the bone formation promoting material C1, the bone fragments were completely ossified, and osteoblasts were not observed.

[ example 4]

A1 mm X3 mm bone defect was made on the femur of a mouse using a drill to obtain a femur defect mouse. The bone formation promoting material 1 obtained in example 1 was transplanted into the femoral defect portion of the obtained femoral defect mouse. The femoral parts of mice 2 days, 7 days, 9 days, 14 days and 21 days after the transplantation were incised to observe the bone formation. In the mouse into which bone formation promoting material 1 was transplanted, the defect portion was subjected to bone healing. The bone formation-promoting material 1 of the present invention significantly promotes bone formation as compared with the conventional bone healing.

[ test example 2] femoral regeneration test

Using the femur-deficient mice, a femoral regeneration test was performed by the following procedure. After the external fixation, the femoral defect was subjected to X-ray imaging and CT imaging 56 days later, and the femoral regeneration performance was evaluated. The conditions for X-ray imaging and CT imaging are shown below. Further, the bone regeneration amount is calculated from the CT imaging result. Of these, 4 mice were used for each bone formation promoting material.

(1) Making model of femur defect

Pentobarbital (manufactured by cochinchinese pharmaceutical corporation) 0.1ml to 0.15ml was injected into the abdominal cavity of a female rat (obtained from china scientific resources) of 10 weeks old to perform bruise, and then the femoral part was cut and an external fixator was attached to the femur (fig. 4). Then, the medial femur of the portion to which the external fixator was attached was cut with a high-speed micro air burr (airtome), and a bone defect of about 5mm was prepared.

(2) Preparation of bone chips

Femurs harvested from the rats were cut with a high-speed micro-air burr (airtome) and bone chips (born chip) were harvested. The bone slices were stored at-80 ℃ prior to use.

(3) Preparation of bone formation promoting material

0.05g of the above bone pieces were mixed with 80. mu.L of self-assembling peptide Gel 1 (trade name: Panacet Gel SPG-178-208, manufactured by Menicon, peptide concentration: 0.8 w/w%) to obtain a bone formation promoting material. Except that self-assembling peptide gel C1(Product name "Puramatrix" manufactured by 3D matrix^TM") or physiological saline was used in place of the self-assembling peptide gel 1, and a bone formation promoting material was obtained in the same manner.

(4) External fixation of femoral defect

Each of the bone formation promoting materials prepared in the above (3) was injected into a protector made of PEEK (manufactured by Yasojima proceedCo., Ltd., length: 5mm, outer diameter: 5mm, inner diameter: 3mm, FIG. 5). Next, a cage into which a bone formation promoting material was injected was inserted into the femoral defect of the femoral defect mouse, and external fixation was performed as shown in fig. 6.

< X-ray imaging >

An X-ray imaging device: SOFTEX/CMB-2 (manufactured by Softex corporation)

(conditions for shooting)

Voltage: 50KVp

Current of 10mA

Time: 15 seconds

Shooting distance: 60cm

Film making: FUJIFILM

< CT imaging >

A CT imaging device: high resolution in vivo micro X-ray CT scanner (SKYSCAN 1176) (shooting condition)

A filter: cu + AL

Power supply voltage: 80KV

Power supply current: 313 muA

Number of rows: 1336

Number of columns: 2000

Image pixel size: 17.6 μm

(reconstitution conditions)

And (3) reconstruction program: NReco

Pixel size 17.60223 μm

CS image conversion minimum: 0.002

CS image conversion maximum: 0.03

< calculation of bone regeneration quantity >

Bone morphology analysis was performed using numerical analysis software "CTAn" manufactured by SKYSCAN corporation, and the amount of bone regeneration was quantified.

(results)

Fig. 7 shows an X-ray photograph and fig. 8 shows a CT photograph of a femoral defect portion externally fixed with each bone formation promoting material. In the mice in which the bone formation promoting material 1 obtained in example 1 was applied to the femoral defect portion, bone regeneration was confirmed in the entire defect portion (SPG-178 in fig. 7 and 8). The bone regeneration amount was 50% or more in all mice (average bone regeneration amount: 66.68%, p: 0.01). On the other hand, in the case of mice to which the bone formation promoting material C1 was applied, bone regeneration was confirmed only from both ends of the femur (PuraMatrix in fig. 7 and 8). In addition, the amount of bone regeneration was small as compared with the mice using the bone formation promoting material 1 (average value of the amount of bone regeneration: 31.54%, p: 0.01). Furthermore, the amount of bone regeneration was small compared to the mice using physiological saline as a control (average value of bone regeneration amount: 44.18%, p ═ 0.01). The amount of bone regeneration in mice using the bone formation promoting material 1 was significantly higher than that in mice using the bone formation promoting material C1.

[ example 5]

Self-assembling peptide gel 3 was diluted with Milli-Q water to give a 1.0 w/w% aqueous solution. The resulting aqueous solution was dropped on a 24-well plate, and the properties of the gel were observed with a stereo microscope (magnification: 100 times). In addition, the pH of the gel was measured using a pH paper.

After 5mg of the bone fragment (rat femur) obtained in test example 2 was put in a test tube, 5. mu.L of the obtained aqueous solution was added dropwise. Subsequently, the mixture was stirred by a vortex mixer, and then air bubbles were removed by a centrifugal separator to obtain a bone formation promoting material 5. The properties of the obtained bone formation promoting material 5 were observed by a stereomicroscope (magnification: 100 times). In addition, the pH of the obtained bone formation promoting material was measured using pH paper.

[ example 6]

A bone formation promoting material 6 was obtained in the same manner as in example 5, except that the self-assembling peptide gel 4 was used. The composition of self-assembling peptide gel 4 is shown in table 1. The properties and pH of the gel before adding the bone chips and the properties and pH of the bone formation promoting material 6 were evaluated in the same manner as in example 5.

Comparative example 2

A bone formation promoting material C2 was obtained in the same manner as in example 5, except that the self-assembling peptide gel C1 was used. The properties and pH of the gel before adding bone chips and the properties and pH of the bone formation promoting material C2 were evaluated in the same manner as in example 5.

[ evaluation ]

Fig. 9 shows photographs of the gels before adding the bone fragments of examples 5 to 6 and comparative example 2, and fig. 10 shows a photograph of a gel (bone formation promoting material) after adding the bone fragments. As shown in fig. 9, the gels before bone chips addition were all homogeneous gels (homogeneous). The bone formation promoting materials of examples 5 and 6 were observed with a stereomicroscope, and as a result, they were in a homogeneous state in which bone fragments were uniformly dispersed (fig. 10(a) and (b)). On the other hand, in the bone formation promoting material of comparative example 2, it was confirmed that the precipitates in the gel state were not uniform (inhomogeneous) (fig. 10 (c)).

In examples 5 and 6, the pH was neutral (about pH 6-8) at any time before and after the addition of the bone fragments. On the other hand, in comparative example 2, the pH of the gel before the addition of the bone chips was acidic (about pH 4), but after the addition of the bone chips, the pH became neutral (about pH6 to 8).

In examples 5 and 6, the pH of the gel was maintained before and after the addition of the bone chips, and thus the obtained bone formation promoting material maintained uniform properties. In these examples, since the bone fragments are uniformly dispersed, it is considered that the bone formation rate is increased by using the bone formation promoting material as a scaffold. On the other hand, in comparative example 2, since the gel was in a heterogeneous state due to a change in pH caused by the addition of the bone fragments, it was considered that the bone formation could not be sufficiently promoted.

Industrial applicability

The bone formation promoting material of the present invention can be advantageously used in the fields of research and development and medical treatment.

Sequence listing

<110> national university legal person famous ancient house university

MENICON Co.,Ltd.

<120> bone formation promoting material

<130>MNC14038PCT

<150>JP2014-134730

<151>2014-06-30

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Claims (7)

1. A bone formation promoting material characterized by:

the self-assembly peptide contains self-assembly peptide and bone fragments which can form a beta sheet structure in a neutral pH aqueous solution, and the sum of the charges of amino acid residues forming the self-assembly peptide at pH7.0 is not 0.

2. The bone formation promoting material as set forth in claim 1, wherein:

the self-assembly peptide is a self-assembly peptide containing the following amino acid sequence,

amino acid sequence: a is₁b₁c₁b₂a₂b₃db₄a₃b₅c₂b₆a₄

In the amino acid sequence, a₁～a₄Is a basic amino acid residue; b₁～b₆Are uncharged polar amino acid residues and/or hydrophobic amino acid residues, and at least 5 of which are hydrophobic amino acid residues; c. C₁And c₂Is an acidic amino acid residue; d is a hydrophobic amino acid residue.

3. The bone formation promoting material as set forth in claim 1 or 2, wherein:

also contains blood and/or blood-derived components.

4. The bone formation promoting material according to any one of claims 1 to 3, wherein: also contains bone formation factor.

5. The bone formation promoting material according to any one of claims 1 to 4, wherein: which is used together with a holding tool for a bone formation promoting material.

6. A holder for a bone formation promoting material as defined in any one of claims 1 to 5.

7. The holder for a bone formation promoting material as set forth in claim 6, wherein:

it is composed of a carbon-based material, an engineering resin or a super engineering resin.