JP2015144767A - Bone formation promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a bone formation promoter that makes it easy to fill the whole of an administration target site with the bone formation promoter reliably.SOLUTION: A bone formation promoter comprises a self-assembly peptide and water, is capable of reversible sol-gel transition under the condition of 20°C-40°C, and has pH of 5.0-8.0.

Description

  The present invention relates to an osteogenesis promoting material containing a self-assembling peptide.

  As a treatment method for bone damage, a method of waiting for the natural regeneration and healing of the living body itself using an artificial bone such as autologous bone or titanium / hydroxyapatite, or mechanical replacement of bone tissue for functional recovery. A method is used. However, the former has a burden on the patient at the time of autologous bone collection, and the latter has problems such as strength and biocompatibility.

  On the other hand, as a technique for actively promoting bone regeneration biologically, attempts have recently been made to transplant cultured cells such as osteoblasts into the affected area, or to place a cell scaffold in the affected area. As the cell scaffold, for example, an extracellular matrix typified by gelatin and collagen is used. However, gelatin and collagen have a drawback that their use is limited by an animal or the like as a source of the material.

  On the other hand, promotion of bone regeneration has been reported in a single administration of a completely synthetic self-assembling peptide or a mixed administration with a bone differentiation-inducing factor (Patent Document 1, Patent Document 2, Non-Patent Document 1). However, osteogenesis promoting materials containing self-assembling peptides, which have been reported to promote bone regeneration, have been applied to every part of the site to be administered that has a complicated or narrow damage shape under neutral conditions. It may be difficult to fill uniformly.

JP 2012-82180 A Special table 2010-504972

Cell Transplantation, Vol. 15, p903-910, 2006

  An object of the present invention is to provide an osteogenesis promoting material that can be easily filled with a reliable osteogenesis promoting material to the entire administration target site.

Since the bone formation promoting material containing a self-assembling peptide that has been confirmed to promote bone regeneration so far is acidic in a solution or sol state, pH substitution is performed so that it becomes a neutral pH, After irreversibly gelling, it is administered to the administration site. On the other hand, when using an osteogenesis promoting material capable of sol-gel transition under normal temperature and neutral pH conditions, the present inventors inject the osteogenesis promoting material into the administration target site in a sol state. It can be gelled, so that even if the administration target site has a complicated or narrow damage shape, reliable filling and retention of the bone formation promoting material to the entire administration target site Has been found to be easily performed, and the present invention has been completed.
According to the present invention, bone formation comprising a self-assembling peptide and water, capable of reversible sol-gel transition under conditions of 20 ° C. to 40 ° C., and having a pH of 5.0 to 8.0. Promotional material is provided.
In one embodiment, in the sol-gel transition, the sol-to-gel phase transition is performed by self-assembly of the self-assembling peptide, and the gel-to-sol phase transition causes physical stimulation of the gel. Done by adding.
In one embodiment, the self-assembling peptide is a self-assembling peptide having a polar amino acid residue and a non-polar amino acid residue, and an acidic amino acid residue and a basic amino acid residue as the polar amino acid residue In the neutral region, the sum of the charge of the acidic amino acid residue and the charge of the basic amino acid residue is a number excluding 0, and only the nonpolar amino acid residue when self-assembled in an aqueous solution Is a self-assembling peptide capable of forming a β-sheet structure arranged on one side.
In one embodiment, the self-assembling peptide is a self-assembling peptide consisting of the following amino acid sequence.
Amino acid sequence: a ₁ b ₁ c ₁ b ₂ a ₂ b ₃ db ₄ a ₃ b ₅ c ₂ b ₆ a ₄
(In the amino acid sequence, a _{1 to} a ₄ are basic amino acid residues; b _{1 to} b ₆ are uncharged polar amino acid residues and / or hydrophobic amino acid residues, provided that at least 5 are hydrophobic amino acid residues; c ₁ and c ₂ are acidic amino acid residues; d is a hydrophobic amino acid residue.)
In one embodiment, the osteogenesis promoting material further includes a liquid medium component.
In one embodiment, the osteogenesis promoting material further includes a physiologically active substance.
In one embodiment, the osteogenesis promoting material further includes cells.
In one embodiment, the osteogenesis promoting material is administered to the administration target site in the form of a solution or a sol, and gels at the administration target site.

  ADVANTAGE OF THE INVENTION According to this invention, the bone formation promotion material which is easy to be reliably filled with the bone formation promotion material to the whole administration object site | part is provided.

It is a graph which shows a time-dependent change of storage elastic modulus. It is a graph which shows a time-dependent change of storage elastic modulus. It is a photograph which shows the filling property to a narrow space. It is a photograph of the bone damage site | part 21 days after injection | pouring of an osteogenesis promoter. It is a graph which shows the bone regeneration volume in the bone damage site | part 21 days after injection | pouring of an osteogenesis promoter.

[A. Bone formation promoter]
The osteogenesis promoting material of the present invention contains a self-assembling peptide and water. The osteogenesis promoting material of the present invention may further contain a physiologically active substance, cells, and / or liquid medium components depending on the application.

  The bone formation promoting material of the present invention has a pH of 5.0 to 8.0, preferably 5.5 to 7.5, and more preferably 6.0 to 7.0. When the pH is within the range, an originally intended intermolecular interaction can occur between the self-assembled peptides, and as a result, a reversible sol-gel transition is possible within the pH range. Moreover, since hydrolysis of the self-assembling peptide during heating can be avoided, it can be subjected to a sterilization process involving heating such as high-pressure steam sterilization. Furthermore, cytotoxicity can be reduced. The pH of the osteogenesis promoter can be measured in the form of a solution or sol.

  The osteogenesis promoter of the present invention can reversibly undergo a sol-gel transition under conditions of 20 ° C to 40 ° C. If the sol-gel transition is possible under the temperature condition, for example, the bone formation promoting material is used as a sol in vitro at room temperature, and the bone formation promoting material in the form of the sol is filled in the administration target site in vivo. Gelation can be performed in a temperature environment (in the case of humans, usually 37 ° C.). The osteogenesis promoting material of the present invention is only required to be capable of sol-gel transition at least at 20 ° C. to 40 ° C., and is less than 20 ° C. (for example, 4 ° C. or more and less than 20 ° C.) or more than 40 ° C. (for example, Sol-gel transition may be possible reversibly even under conditions of more than 40 ° C. and not more than 50 ° C.).

  Regarding the explanation of the sol-gel transition, “sol” is a colloid having fluidity. In one embodiment, the sol is more fluid than the solution. The bone formation promoting material that is a sol typically includes a fibrous molecular assembly of self-assembled peptides as a dispersoid. On the other hand, “gel” is a sol solidified in a jelly form. In an osteogenesis promoting material that is a gel, typically, fibrous molecular aggregates are connected via an interaction to form a three-dimensional network structure. The fibrous molecular assembly and the three-dimensional network structure can be confirmed by observation with a microscope (for example, an electron microscope). In addition, “sol-gel transition” refers to a phenomenon in which a sol is phase-transformed into a gel or a gel is phase-transformed into a sol.

  The storage elastic modulus (G ′) at 37 ° C. in the dynamic viscoelasticity measurement using the rotary rheometer of the bone formation promoting material of the present invention is, for example, 600 Pa or less, preferably 1 Pa to 600 Pa, more preferably 1 Pa. ~ 150 Pa. If the storage elastic modulus is within the above range, the function as a scaffold for bone formation and the permeability that allows cells to enter the gel can be suitably achieved. In addition, a hemostatic effect can be achieved by pressing the administration target site. In addition, the storage elastic modulus as used in this specification means the value when the angular frequency when frequency change measurement is performed is 1 radian / second.

  In the sol-gel transition of the osteogenesis promoter of the present invention, the phase transition from sol to gel is performed by self-assembly of self-assembling peptides. Thus, the gel-form osteogenesis promoter is a self-assembled peptide gel.

  On the other hand, the phase transition from gel to sol is typically performed by applying a physical stimulus to the gel. The physical stimulus can be any suitable stimulus as long as the gel can undergo a phase transition to the sol. Specific examples of the physical stimulus include vibration, stirring, pressure change and the like. More specifically, stimulation that can apply a shearing force to the gel, such as pipetting; vortexing; sonication; extrusion from a pipette, tube, syringe, or the like;

[Self-assembled peptide]
As the self-assembled peptide, any suitable peptide that can spontaneously assemble through the interaction of peptide molecules in an aqueous solution to form a gel can be used. More specifically, a fibrous molecular assembly is formed spontaneously through interaction between peptide molecules in an aqueous solution, and a three-dimensional network structure is developed by the interaction between the molecular assemblies. Peptides that can form gels can be preferably used. Examples of the interaction between peptide molecules include electrostatic interactions such as hydrogen bonds, ionic interactions, van der Waals forces, and hydrophobic interactions.

  The amino acid constituting the self-assembling peptide may be an L-amino acid or a D-amino acid. L-amino acids are preferred. Moreover, a natural amino acid may be sufficient and a non-natural amino acid may be sufficient. Natural amino acids are preferred because they are available at low cost and facilitate peptide synthesis.

  The sum of the charges at pH 7.0 of the amino acid residues constituting the self-assembling peptide is preferably not 0. The total sum of the charges is more preferably −3 to −1 or +1 to +3, and further preferably −3, −2, +2 or +3. Thus, the balance between the electrostatic attractive force and the repulsive force suitable for gel formation can be obtained because the positive charge and the negative charge derived from the side chain of the amino acid residue contained in the self-assembling peptide are not offset in the neutral region. This is because, as a result, a transparent and stable gel can be formed in the neutral region. In the present specification, the “neutral region” means pH 5.0 to 8.0, preferably pH 5.5 to 7.5, more preferably pH 6.0 to 7.0, and further preferably pH 7.0. Refers to the area.

  The charge of the self-assembling peptide at each pH can be calculated, for example, according to the method of Lehninger (Biochimie, 1979). The Rainer's method can be performed, for example, by a program available on the EMBL WWW Gateway to Isoelectric Point Service website (http://www.embl-heidelberg.de/cgi/pi-wrapper.pl).

Specific examples of the self-assembling peptide that can be preferably used in the present invention include peptides having the amino acid sequence of the following formula (I).
a ₁ b ₁ c ₁ b ₂ a ₂ b ₃ db ₄ a ₃ b ₅ c ₂ b ₆ a ₄ (I)
(In the amino acid sequence, a _{1 to} a ₄ are basic amino acid residues; b _{1 to} b ₆ are uncharged polar amino acid residues and / or hydrophobic amino acid residues, provided that at least 5 are hydrophobic amino acid residues; c ₁ and c ₂ are acidic amino acid residues; d is a hydrophobic amino acid residue.)

In the amino acid sequence, a _{1 to} a ₄ are basic amino acid residues. 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 _{1 to} a ₄ may be the same amino acid residue or different amino acid residues.

In the amino acid sequence, b _{1 to} b ₆ are uncharged polar amino acid residues and / or hydrophobic amino acid residues, and at least five of them 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, glutamine, or cysteine. This is because these amino acids are easily available.

Preferably, b ₃ and b ₄ are each independently any suitable hydrophobic amino acid residue, more preferably a leucine residue, an alanine residue, a valine residue, or an isoleucine residue, particularly preferably Is a leucine residue or an alanine residue.

Preferably, b _{1 to} b ₆ are all hydrophobic amino acid residues. This is because the self-assembling peptide preferably forms a β-sheet structure and can self-assemble. More preferably, b _{1 to} b ₆ are each independently 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, 4 or more of b _{1 to} b ₆ are leucine residues, more preferably 5 or more of them are leucine residues, and more preferably all are leucine residues.

In the amino acid sequence, c ₁ and c ₂ are acidic amino acid residues. The acidic amino acid is preferably aspartic acid or glutamic acid. This is because these amino acids are easily available. c ₁ and c ₂ may be the same amino acid residue or different amino acid residues.

  In the 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 one preferred embodiment, two of the three consecutive amino acid residues of b ₃ , d, b ₄ are leucine residues and the rest are alanine residues. In this case, any of b ₃ , d, and b ₄ may be an alanine residue. In another preferred embodiment, all three consecutive amino acid residues of b ₃ , d, and b ₄ are leucine residues.

Preferred specific examples of the amino acid sequence of the formula (I) are exemplified below.
n-RLDLRLALRLLDLR-c (SEQ ID NO: 1)
n-RLDLRLLLLRLDLR-c (SEQ ID NO: 2)
n-RADLRLALRLLDLR-c (SEQ ID NO: 3)
n-RLDLRLALLRLDA-c (SEQ ID NO: 4)
n-RADLRLLLRLLDLR-c (SEQ ID NO: 5)
n-RADLRLLLRLDA-c (SEQ ID NO: 6)
n-RLDLRALLRLLDLR-c (SEQ ID NO: 7)
n-RLDLRLLARLDLR-c (SEQ ID NO: 8)

  Another self-assembling peptide that can be preferably used in the present invention is 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 includes an acidic amino acid residue and a basic amino acid residue, and in the neutral region, the sum of the charge of the acidic amino acid residue and the charge of the basic amino acid residue is 0. And a self-assembling peptide capable of forming a β-sheet structure in which only the nonpolar amino acid residues are arranged on one side when self-assembled in an aqueous solution.

Among the above self-assembling peptides, peptides containing acidic amino acid residues, basic amino acid residues, and uncharged polar amino acids are preferred as polar amino acids. Preferred specific examples of such self-assembling peptides are exemplified below.
n-RASARADARASARADA-c (SEQ ID NO: 9)
n-RANARADARANARADA-c (SEQ ID NO: 10)
n-RAAARADAARAAARADA-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 a chemical synthesis method such as a solid phase method such as the Fmoc method or a liquid phase method, and a molecular biological method such as gene recombinant expression.

  The self-assembling peptide may be subjected to any appropriate modification depending on the purpose and the like. The site where the modification is performed is not particularly limited, and examples thereof include an N-terminal amino group, a C-terminal carboxyl group, or both of the self-assembling peptide.

  As the modification, any appropriate modification can be selected as long as the modified peptide has the ability to self-assemble. For example, introduction of protecting groups such as acetylation of N-terminal amino group and amidation of C-terminal carboxyl group; introduction of functional groups such as alkylation, esterification or halogenation; hydrogenation; monosaccharide, disaccharide, oligo Introduction of sugar compounds such as sugars or polysaccharides; introduction of lipid compounds such as fatty acids, phospholipids or glycolipids; introduction of amino acids or proteins; introduction of DNA; introduction of compounds having other physiological activities. Only one type of modification may be performed, or two or more types may be combined. For example, the N-terminus of an added peptide having a desired amino acid introduced at the C-terminus of the self-assembling peptide may be acetylated and the C-terminus amidated.

  When an amino acid or protein is introduced, the number of amino acids to be introduced 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. ~ 5. If the number of amino acid residues to be introduced exceeds 180, the self-organizing ability may be impaired.

  The osteogenesis promoting material of the present invention may contain only one kind of self-assembling peptide, or may contain two or more kinds of self-assembling peptides.

  The concentration of the self-assembling peptide in the osteogenesis promoting material of the present invention can be appropriately set depending on the composition, use, and the like. The concentration of the self-assembling peptide is preferably 0.1% to 5.0% by weight, more preferably 0.1% to 1.5% by weight, and still more preferably 0.2% to 1.0% by weight. %. If it is the density | concentration of the said range, the effect of this invention can be acquired suitably.

[water]
As the water, purified water such as ion-exchanged water or distilled water can be preferably used.

  The moisture content (%) of the osteogenesis promoting material of the present invention (= weight of water in osteogenesis promoting material / total weight of osteogenesis promoting material × 100) is, for example, 80% to 99.9%, preferably 85%. It can be ˜99.8%.

[cell]
The cells that can be included in the osteogenesis promoting material of the present invention can be any appropriate cells depending on the application and the like. Specific examples of cells include mesenchymal stem cells such as dental pulp stem cells, universal cells such as ES cells and iPS cells, osteoclasts, osteoblasts and other cells directly involved in bone formation and their precursor cells, bone formation Cells involved in the differentiation and functional expression of the cells involved, cells involved in immune / inflammatory functions, cells controlling cell migration and / or division, cells synthesizing / secreting extracellular matrix, and Examples thereof include cells that perform the control. These cells may be primary cells directly collected from tissues or organs, may be subcultured from them, or may be preserved (freeze preserved, cryopreserved). The cells may also be cultured in the osteogenesis promoting material for a predetermined period, or may be differentiated or dedifferentiated into other cells during the culture period. The culture period varies depending on the administration subject, cell type, and the like, but may be, for example, about 1 to 20 days.

  The origin of the cell can be, for example, human, dog, cat, rabbit, rat, mouse, pig, sheep, monkey and the like.

[Bioactive substances]
Examples of the physiologically active substance that can be included in the osteogenesis promoting material of the present invention include differentiation regulators that induce or promote differentiation of bone or cartilage (for example, TGF-β); osteogenic factors; growth hormone; cells such as EGF and FGF Function control factors; immune or inflammation-related factors such as interferon and interleukin; Specific examples of osteogenic factors include BMP families such as BMP-2, BMP-4, BMP-6, BMP-7 and BMP-9. Since these human BMPs have already been cloned, they can be obtained by genetic engineering techniques based on their nucleotide sequences.

  The concentration of the physiologically active substance in the bone formation promoting material of the present invention is usually 0.0001 ppm to 1000 ppm, preferably 0.01 ppm to 1000 ppm. If it is the said density | concentration range, the effect | action of a bioactive substance will be exhibited suitably and bone formation may be accelerated | stimulated.

[Other additives]
The osteogenesis promoting material of the present invention may further contain any appropriate additive as required. Specific examples of the additive include pH adjusters; buffers; isotonic agents; salts; amino acids; vitamins; alcohols; cell culture medium components; These additives may be used alone or in combination of two or more.

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

  Buffers include phosphates such as phosphoric acid, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate; boric acid, borax Borate salts such as sodium borate and potassium borate; citrate salts such as sodium citrate and disodium citrate; acetates such as sodium acetate and potassium acetate; Tris and HEPES.

  Isotonic 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; mannitol, sorbitol and the like Sugar alcohols; and the like.

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

  Any appropriate liquid medium component can be selected as the medium component for cell culture depending on the purpose and the like. Examples of the liquid medium include eagle medium (for example, MEM, DMEM, MEMα) and the like.

  The addition amount of the additive can be set to any appropriate value depending on the purpose and the like.

[B. Preparation method]
The method for preparing an osteogenesis promoting material of the present invention includes mixing a self-assembling peptide, water, and optional components. Thereby, the osteogenesis promoting material in the form of a solution can be obtained. The mixing order is not particularly limited, and is appropriately set according to the purpose and the like. For example, from the viewpoint of uniformly dispersing or dissolving the self-assembling peptide, components that are unsuitable for vigorous stirring such as cells can be added after mixing the self-assembling peptide and water.

  If necessary, an osteogenesis promoting material in the form of a solution obtained by mixing the self-assembling peptide, water, and optional components is allowed to stand. As a result, the self-assembled peptide spontaneously aggregates in the solution through the interaction between the peptide molecules to form a fibrous molecular aggregate, which is in the form of a sol. Furthermore, by continuing to stand still, a three-dimensional network structure develops by the interaction between the said molecular assemblies, and it becomes a gel form. The standing time and the standing temperature can be appropriately set according to the administration subject, the concentration and type of the self-assembling peptide, and the like.

  The method for preparing the osteogenesis promoting material of the present invention may further include optional steps such as purification such as filtration; sterilization such as high-pressure steam sterilization, radiation sterilization, and dry heat sterilization;

  In one embodiment, a method for preparing an osteogenesis promoting material includes, for example, mixing a self-assembling peptide, water, a cell culture medium component, and any other components, and then adding cells to the resulting mixture. (For example, osteoblasts, dental pulp stem cells, etc.) may be added and suspended, and the resulting cell suspension may be allowed to stand and gel. If necessary, the obtained gel may be further placed under conditions suitable for cell culture, and cells may be cultured. Conditions suitable for cell culture can be appropriately set by those skilled in the art depending on the cell type.

  According to the preparation method including the cell culture step, an osteogenesis promoting material containing cells embedded and cultured in a gel can be obtained. Since the cultured cells can be in a differentiation-induced state or in a state suitable for proliferation, good bone formation can be expected by administering a bone formation promoting material containing cells in such a state.

[C. how to use]
The osteogenesis promoting material of the present invention is preferably administered to a site to be administered in the form of a solution or a sol, and gelled and placed at the site to be administered. From the viewpoint of filling every corner of a complex or narrow administration target site (for example, a gap between bone cracks) and reducing leakage, it is preferably administered in the form of a sol and gelled at the administration target site. Of course, a bone formation promoting material in the form of a gel may be administered (injected) to the administration target site.

  In one embodiment, the method of using the osteogenesis promoting material of the present invention comprises preparing an osteogenesis promoting material in the form of a solution or sol, and injecting the osteogenesis promoting material in the form of a solution or sol into the administration target site. And gelling the bone formation promoting material at the site to be administered.

  A solution-form osteogenesis promoter can be obtained by mixing its constituents. On the other hand, the bone formation promoting material in the sol form allows the bone formation promoting material in a solution form to stand to advance the self-assembly of the self-assembling peptide, or the bone formation promoting material once gelled is physically stimulated. Can be obtained. In conventional osteogenesis promoting materials, for example, as described in paragraph 0171 of Patent Document 2, when neutral pH is set, the neutral sol is rapidly and irreversibly gelled. It was very difficult to administer. On the other hand, the osteogenesis promoting material of the present invention has a neutral pH and is capable of reversible sol-gel transition under a predetermined temperature condition, and therefore is in a physiological environment in the form of a sol. It can be administered to the site of administration, gelled on the spot, and placed.

  As the physical stimulus, a stimulus to such an extent that the fibrous molecular aggregate forming the gel is maintained is preferable. For example, ultrasonic irradiation, pipetting, vortexing and the like can be mentioned.

  Injection of the osteogenesis promoting material into the administration target site can be performed using any appropriate means such as a syringe, a tube, and a pipette.

  Since the administration target site is usually in a physiological environment, gelation of the osteogenesis promoting material of the present invention at the administration target site can proceed spontaneously.

  Since the osteogenesis promoting material of the present invention is excellent in biodegradability, it can be decomposed or absorbed over time with bone formation at the administration target site.

  The bone formation promoting material of the present invention can be used, for example, for repairing a defect site in bone tissue, periodontal tissue, and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited by these Examples.

[Test Example 1: Evaluation of sol-gel transition ability]
A self-assembling peptide solution (SPG-178 (1 wt% solution of peptide of SEQ ID NO: 1 with N-terminal acetylated and C-terminal amidated), pH 6.60) was allowed to gel at room temperature. Was used as Sample 1. In addition, 300 μL of self-assembling peptide solution (manufactured by 3D Matrix, product name “Puramatrix ^™ ”, 1 wt% solution of Ac-RADARADARADARADA-CONH ₂ ) in 1% Na ₂ CO ₃ -HCl buffer (pH 6.82) 1000 μL And the supernatant was removed, and the remaining gel mass was used as Sample 2. In addition, pH measured the pH of the sample of a solution form using the portable pH meter (Horiba Ltd. make, product number "B-712").

Samples 1 and 2 were mechanically sheared, and the change in storage modulus immediately after shearing was measured over time. The measuring method of storage elastic modulus is as follows.
≪Measurement of storage modulus 1≫
The storage elastic modulus G ′ was measured using a rotary rheometer (manufactured by TA instruments, product name “ADVANCED RHEOMETER AR 1000”) which is a dynamic viscoelasticity measuring apparatus. Specifically, it is as follows. First, the geometry (aluminum cone, diameter 20 mm, cone angle 1 °, gap 24 μm) for contacting the sample and a thermostat for maintaining the sample stage at a constant temperature were attached to the rheometer. Subsequently, the measurement was performed according to the following procedure under measurement conditions of temperature: 37 ° C., torque: 1 μN · m, and frequency: 1 rad / s. The outline of the measurement is that a physical force is applied to the gel by pre-shearing and then the change in storage modulus over time is observed.
(1) Using a pipette, place 55 μL of sample on the sample stage of the rheometer.
(2) The geometry is moved from the sample stage to a gap of 24 μm and brought into contact with the sample.
(3) Move the geometry slightly to blend with the sample.
(4) A sorbent trap is attached to prevent the solvent from evaporating from the sample.
(5) Pre-shearing is performed by rotating the geometry at 10 Pa or 100 Pa for 30 seconds.
(6) The storage modulus is measured over time at intervals of about 20 seconds.

  As a result of the pre-shearing, the gel of Sample 1 became a colorless and transparent fluid, and then left to stand at room temperature, whereby the elastic storage rate increased over time and gelled again (see FIG. 1). From this, it can be seen that Sample 1 is capable of reversible sol-gel transition under normal temperature neutral conditions. On the other hand, the gel of sample 2 was disintegrated by pre-shearing, and became a suspended state of fine gel. Thereafter, it was allowed to stand at room temperature, but no increase in elastic storage rate was observed (see FIG. 2). From this, it can be seen that Sample 2 cannot reversibly undergo a sol-gel transition under normal temperature neutral conditions.

[Test Example 2: Fillability evaluation]
A self-assembling peptide solution (1% by weight solution of SPG-178 (the peptide of SEQ ID NO: 1 with N-terminal acetylation and C-terminal amidation), pH 6.60) was diluted with an equal amount of pure water, The obtained diluted solution was allowed to stand at room temperature and gelled, and used as Sample 3. In addition, a self-assembling peptide solution (manufactured by 3D Matrix, product name “Puramatrix ^™ ”, 1% by weight solution of Ac-RADARADARADARADA-CONH ₂ ) is diluted with an equal amount of pure water, and the resulting diluted solution (sol) 1000 μL of 1% Na ₂ CO ₃ -HCl buffer (pH 6.84) is left in 300 μL, and after confirming that the whole has gelled, the supernatant is removed and the remaining gel mass is used as sample 4. Using.

Using the samples 3 and 4, a model system was prepared assuming a narrow portion such as a crack caused by bone damage, and the filling property was evaluated as follows.
≪Fillability evaluation≫
Combining biological cover glasses (Matsunami Glass 18 mm × 18 mm) to create a rectangular parallelepiped narrow space (width: 0.9 mm to 1.1 mm, height: 0.12 mm to 0.17 mm) having an opening. . About 50 μL of each of Samples 3 and 4 subjected to shearing treatment by pipetting several times was dropped into the opening, and the filling state after about 20 seconds was observed. The results are shown in FIGS. 3 (a) and (b).

  The gel of Sample 3 became a colorless and transparent sol with high fluidity by shearing treatment, and smooth penetration into the narrow space was observed (see FIG. 3A). On the other hand, the gel of sample 4 collapsed due to the shearing process and became a suspended state of the fine gel, and the liquid phase component began to permeate quickly into the narrow space. It remained outside (see FIG. 3 (b)).

[Test Example 3: Evaluation of relationship between peptide concentration and storage modulus]
A self-assembling peptide (SPG-178) powder is added to pure water and mixed with a rotating and rotating stirrer, and three types of self-assembling peptide solutions having different peptide concentrations (0.4 wt%, 0.8 wt%) % And 1.5% by weight). The obtained self-assembled peptide solution was allowed to stand at room temperature to obtain a self-assembled peptide gel. The storage elastic modulus of the obtained gel was measured as follows. The results are shown in Table 1 together with the pH of the self-assembled peptide solution. The pH of the self-assembled peptide solution was measured using a portable pH meter (manufactured by Horiba, Ltd., product number “B-712”).

≪Measurement of storage modulus 2≫
The storage elastic modulus G ′ of the sample (gel) was measured using a rotary rheometer (manufactured by TA instruments, product name “ADVANCED RHEOMETER AR 1000”) which is a dynamic viscoelasticity measuring apparatus. Specifically, it is as follows. First, the geometry (aluminum cone, diameter 20 mm, cone angle 1 °, gap 24 μm) for contacting the sample and a thermostat for maintaining the sample stage at a constant temperature were attached to the rheometer. Next, under the measurement conditions of temperature: 37 ° C., torque: 1 μN · m, frequency: 0.5 rad / s to 100 rad / s, a sample was measured three times by the following measurement procedure, and the value at 1 rad / s. Was the storage elastic modulus G ′.
(1) Using a pipette, place 55 μL of sample on the sample stage of the rheometer.
(2) The geometry is moved from the sample stage to a gap of 24 μm and brought into contact with the sample.
(3) Move the geometry slightly to blend with the sample.
(4) Measurement is started 15 seconds after stopping the movement of the geometry (a solvent trap for preventing volatilization of the solvent from the sample is attached during the 15 seconds).

[Example 1]
A gel-like osteogenesis promoter 1 was obtained by mixing a 0.4 wt% aqueous solution of a self-assembling peptide (SPG-178) and a MEMα medium at 1: 1 (volume ratio) and allowing to stand.

[Example 2]
A self-assembling peptide (SPG-178) 0.4% by weight aqueous solution and 0.0001% (w / v) BMP-4 (manufactured by R & D systems, product number “314-BP”)-containing MEMα medium were 1: 1. The gel-like bone formation promoting material 2 was obtained by mixing by (volume ratio) and leaving still.

[Example 3]
0.0001% (w / v) BMP- in which a 0.4 wt% aqueous solution of self-assembling peptide (SPG-178) and SD rat-derived self-adjusted rat dental pulp stem cells are suspended at a concentration of 4.0 × 10 ⁶ cells / mL 4 (R & D systems, product number “314-BP”)-containing MEMα medium was mixed at a ratio of 1: 1 (volume ratio) and allowed to stand to obtain a gel-like bone formation promoting material 3.

Regarding the bone formation promoting materials 1 to 3, the bone repairing action was evaluated. Specifically, it is as follows.
(Production of bone damage model)
After general anesthesia of 4-5 week old SD rats (CLEA Japan), the skin of the parietal region was incised, and the center of the skull was crossed over the midline using the dental implant bar 5.0 (manufactured by Technica). One through hole with a diameter of 5 mm was created per individual.

(Injection of bone formation promoter into bone damage site)
At room temperature, the bone formation promoting material 1 was made into a sol by pipetting several times. 70 μL of the obtained sol was injected into the bone damage site. Since the sol has high fluidity, it was easy to fill every part of the bone damage site, and gelled within several minutes after the filling and was fixed to the bone damage site. After confirming gelation, the skin was sutured. After confirming the awakening of anesthesia, it was transferred to a predetermined animal breeding facility and reared for 21 days. Bone damage promoting materials 2 and 3 were similarly prepared using a separate body and filled with bone damage. In addition, after creating a bone damage site, a test section similar to that in Example 1 was provided except that skin suture was performed without filling anything, and was used as a control.

(Repair degree evaluation)
21 days after injection of the osteogenesis promoting material into the bone injury site, euthanasia treatment was performed by CO ₂ treatment. After the skull was removed, the bone regeneration volume was measured using a 3D micro CT imaging apparatus for laboratory animals “R-mCT (Rigaku)”. FIG. 4 shows a CT scan image of a bone injury site 21 days after the injection. Moreover, the bone regeneration volume in a bone damage site | part is shown in FIG.

(result)
As is clear from FIGS. 4 and 5, the osteogenesis promoting materials 1 to 3 of the present invention all showed an excellent osteogenesis promoting action.

  The osteogenesis promoting material of the present invention can be suitably used in the fields of research and development and medicine.

Claims (8)

  An osteogenesis promoter comprising a self-assembling peptide and water, capable of reversible sol-gel transition at 20 ° C to 40 ° C, and having a pH of 5.0 to 8.0.   In the sol-gel transition, a sol-to-gel phase transition is performed by self-assembly of the self-assembled peptide, and a gel-to-sol phase transition is performed by applying a physical stimulus to the gel. The bone formation promoting material according to claim 1. The self-assembling peptide is a self-assembling peptide having a polar amino acid residue and a nonpolar amino acid residue,
Including acidic amino acid residues and basic amino acid residues as the polar amino acid residues,
In the neutral region, the sum of the charge of the acidic amino acid residue and the charge of the basic amino acid residue is a number excluding 0,
The osteogenesis promoting material according to claim 1 or 2, which is a self-assembling peptide capable of forming a β-sheet structure in which only the nonpolar amino acid residues are arranged on one side when self-assembling in an aqueous solution. . The osteogenesis promoting material according to claim 1 or 2, wherein the self-assembling peptide is a self-assembling peptide having the following amino acid sequence.
Amino acid sequence: a ₁ b ₁ c ₁ b ₂ a ₂ b ₃ db ₄ a ₃ b ₅ c ₂ b ₆ a ₄
(In the amino acid sequence, a _{1 to} a ₄ are basic amino acid residues; b _{1 to} b ₆ are uncharged polar amino acid residues and / or hydrophobic amino acid residues, provided that at least 5 are hydrophobic amino acid residues; c ₁ and c ₂ are acidic amino acid residues; d is a hydrophobic amino acid residue.)   The bone formation promoting material according to any one of claims 1 to 4, further comprising a liquid medium component.   The bone formation promoting material according to any one of claims 1 to 5, further comprising a physiologically active substance.   The bone formation promoting material according to any one of claims 1 to 6, further comprising cells.   The osteogenesis promoting material according to any one of claims 1 to 7, which is administered to a site to be administered in the form of a solution or a sol and gels at the site to be administered.