# Patent

## Title
Growth factor anchoring type bone graft material, method for producing growth factor anchoring type bone graft material, kit for producing growth factor anchoring type bone graft material, and method for forming bone

## Abstract

Provided is a growth factor anchoring type bone graft material, wherein a bone graft substrate exposing at least a collagen fiber is bound to a collagen-binding-site-containing growth factor which contains a growth factor receptor agonist peptide and a collagen-binding peptide. The same can be produced by mixing a bone graft substrate and a collagen-binding-site-containing growth factor which contains a growth factor receptor agonist peptide and a collagen-binding peptide, and is also superior in osteogenic ability.

## Background
TECHNICAL FIELD
The present invention relates to a bone graft material bound a bone graft substrate exposing at least a collagen fiber to a growth factor, more particularly, relates to a growth factor anchoring type bone graft material wherein a bone graft substrate is bound to a collagen-binding-site-containing growth factor which comprises a growth factor receptor agonist peptide and a collagen-binding peptide, a method for producing the growth factor anchoring type bone graft material, a kit for production of a growth factor anchoring type bone graft material, and a method for forming a bone.
BACKGROUND ART
When an artificial joint has been implanted for treating articular rheumatism or arthrosis deformans and caused loosening between the artificial joint and bone tissues after long period service, it should be replaced by a new one through artificial joint revision surgery. On an artificial joint revision surgery, bone grafting with an autologous bone derived from the patient, or the like, is carried out in order to supplement a part of lost bone. Bone grafting has a feature that a bone protein contained in grafted bone promotes resorption of the grafted bone and conversion to an autologous tissue, therefore it has an advantage that reconstruction of a joint function becomes possible even though reconstruction with a prosthesis is impossible. Further, bone is a tissue superior in regenerative capacity, it may be regenerated into a nearly original form by proper reintegration and fixation in case of a fracture.
However, autologous bone grafting is a method which own bone is cut out from a certain part of a patient as a block, the obtained bone is transplanted to deficient part as a block, or after crushing to a granular or powder form. The method is an advantage of high safety because own bone is utilized although, pains are severe at the bone collecting part in the case of a large bone defect region, the recovery period after the bone grafting surgery becomes longer, and sometimes it is very difficult to find a donor supplying a bone for bone grafting. To avoid such drawbacks, allogeneic bone grafting using a donor-derived bone instead of an autologous bone is conducted, and further, various bone graft materials have been also developed.
For example, there is a composition used for promoting bone formation in arthrodesis which includes a platelet-derived growth factor solution, a biocompatible matrix containing polysaccharides, and a scaffold material such as calcium phosphate (Patent Literature 1). In the example thereof, 1.0 mg/mL of platelet-derived growth factor is dropped to calcium phosphate in the average diameter of 1000 to 2000 μm for preparing a composition, and the composition is coated on a bone to be fused in a joint. As the result, the composition exhibits bone bridging and joint adhesion equivalent to autologous bone grafting.
Further, there is a bone graft material on which surface a cell adhesion inducing peptide having an RGD amino acid sequence, or a tissue growth factor-derived peptide is fixed (Patent Literature 2). The bone graft material adhering on the surface a tissue growth factor capable of obtaining a tissue regeneration effect and a peptide having active site of an extracellular matrix protein exhibits allegedly a stable and sustainable pharmacological effect, even though the concentration of the peptides is low. In the example thereof surfaces of a bovine bone-derived bone mineral particle are treated with 3-aminopropyltriethoxysilane to form an amine residue, the particles are bound with a crosslinking agent of 1,4-bis-maleimidebutane added thereto, then reacted with a cell adhesion inducing peptide to fix the peptide, and prepare a bone graft material. The material exhibits allegedly superior regenerative power compared to a bone graft material without the fixed peptide.
There is also a bone graft fragment composition prepared by drying a fragment of a cell-free tissue substrate together with a fragment of a demineralize bone material (Patent Literature 3). A cell-free tissue substrate such as collagen obtained from an epithelial cell has capability for supporting cell recognition and cell association, as well as cell spreading, cell proliferation, and cell differentiation, a demineralize bone material has physiological characteristics of natural bone important for a success of bone grafting. When the obtained bone graft fragment composition is coated on a transplantation or implantation part after hydration, new bone formation can be allegedly induced in or on a surface of an osseous tissue, or in or on a surface of a non-osseous tissue of a recipient by stimulating a bone formation stem cell.
Meanwhile, there is also a composition containing a fusion protein fused a PTH/PTHrP receptor agonist with a collagen-binding polypeptide fragment drived from a collagenase (Patent Literature 4). A parathyroid hormone (PTH) is used for an anabolic therapy of osteoporosis, an administration once a day is required. The composition can form a stable bind with collagen through a collagen-binding polypeptide fragment, and stay at an administration site for a long time period resisting body fluid circulation to enjoy longer half-life than PTH. Then, it can exert allegedly the same or higher effectiveness compared to PTH administration. In the example, it is administered intraperitoneally and increase of the bone density is observed.
Further, a fusion protein which a basic fibroblast growth factor (bFGF) instead of a PTH/PTHrP receptor agonist is bound to a collagen-binding polypeptide fragment, has been also known (Non Patent Literature 1).
Further, based on knowledge that it is useful to use a bone promoting factor in a treatment of a fracture, there is a bone formation promoting fusion protein prepared by binding a polypeptide having a collagen-binding domain derived from fibronectin with a bone formation promoting protein (Patent Literature 5). As examples of the bone formation promoting protein are named a growth factor belonging to a BMP (Bone Morphogenetic Proteins) subfamily, bFGF, and a thyroid hormone. In the example the polypeptide is prepared by using mRNA extracted from human kidney cells as a template thereof, bound with BMP2 or BMP7 as the bone formation promoting protein to prepare the bone formation promoting fusion protein. When the fusion protein was suspended with an osteoblast to be a mouse calvarium-derived established cell, administration of the bone formation promoting fusion protein caused allegedly concentration-dependent enhancement of alkali phosphatase activity on an osteoblast compared to administration of the above polypeptide.
Further, there is a composition for a treatment of a bone defect composed of a forming particle having at least 4 curved projections composed of calcium sulfate or the like and a material for a suspension (Patent Literature 6). A plurality of the projection of the forming particle can interlock each other to stabilize filling into a defect site, a binder capable of forming a gel of a collagen derivative or the like, or a bone morphogenic protein (BMP) can use as the suspension.
Further, there is a self-curing porous calcium phosphate composition which contains calcium phosphate, a blowing agent, and a biocompatible flocculant, and is mixed with a physiologically acceptable liquid, can releases a gas component by hydration of the blowing agent in the composition, gives at least 5% of porosity to the composition, and after curing the calcium phosphate composition exhibits a compressive strength of 1 MPa or more (Patent Literature 7). As the biocompatible flocculant collagen is disclosed and it is described that the composition may contain further a collagen exposure-treated substrate. The invention has a feature that a porous calcium phosphate composition is formed by a blowing agent, and in the example thereof a collagen exposure-treated substrate, sodium hydrogen carbonate and calcium phosphate as a blowing agent, and carboxymethyl cellulose as a flocculant were mixed to prepare a self-curing paste. By filling the self-curing paste in a defect formed at a rabbit distal femoral condyle, nearly complete healing was allegedly observed.
Additionally, there is a bone growth composition containing a particulate fibrous collagen component, and a calcium phosphate component, as well as a substance selected from the group consisting of a purified bone growth factor, a recombinant bone growth factor, a bone-marrow component, and demineralized bone and autologous bone (Patent Literature 8). The collagen component is cross-linked collagen or porous granular or other insoluble collagen. In the example, a calcium phosphate gel dispersion is kneaded with complex collagen, and after a cross-linking step by freeze-drying and thermal dewatering shaped into the particulate, pasted by adding blood, then transplanted to scattered bone. A defect site could be allegedly fixed firmly with the paste.
CITED LITERATURE
Patent Literatures
Patent Literature 1: Japanese National Publication of International Patent Application No. 2010-508912.
Patent Literature 2: Japanese National Publication of International Patent Application No. 2007-530099.
Patent Literature 3: Japanese National Publication of International Patent Application No. 2009-534125.
Patent Literature 4: Japanese National Publication of International Patent Application No. 2010-523671.
Patent Literature 5: Unexamined Japanese Patent Application Kokai Publication No. 2002-58485.
Patent Literature 6: Japanese National Publication of International Patent Application No. 2003-525696.
Patent Literature 7: Japanese National Publication of International Patent Application No. 2009-519052.
Patent Literature 8: Japanese National Publication of International Patent Application No. 2010-512967.
Non Patent Literature
Non Patent Literature 1: “Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain”, Nozomu Nishi, et al., Proc. Natl. Acad. Sci., USA, Vol. 95, pp 7018-7023, June 1998, Medical Sciences.
SUMMARY OF INVENTION
Technical Problem
Bone grafting is exercised on an artificial joint revision surgery, a treatment of fracture, and a bone defect due to malignant osteosarcoma, but in some cases, even though graft bone originated from autologous bone or allogeneic bone is used, a graft bone applied to a bone occurs faulty union or delayed union to the site of application to the bone reportedly. Such faulty union or delayed union means prolongation of a treatment period and becomes an economical, physical, and mental burden on the patient. In view of the situation that fractures occur frequently among aged persons in the recent aging society, soonest bone union is desired in order to initiate rehabilitation as soon as possible.
However, the bone graft material of Patent Literature 1, although calcium phosphate or the like used as a scaffold material is advantageous in terms of easy availability, bone growth or early union surpassing autologous bone is difficult. In the case of the bone graft material of Patent Literature 2, a cell adhesion inducing peptide or a tissue growth factor-derived peptide is fixed on a bone surface, the same can remain at an administration part at a high retention rate, and exhibit superior bone regenerative power. It, however, requires a cross-linking treatment for fixing the peptide on the bone surface, which makes the production difficult. Meanwhile, Patent Literature 3 requires use of a demineralized bone material, and for demineralization extraction with 0.6 N hydrochloric acid for 3 to 24 hours is necessary, namely the treatment time becomes longer. Further, although it is advantageous that the bone graft material of Patent Literature 3 or Patent Literature 4 uses an active ingredient relevant to bone growth, such a component is easy to leave from the administrated part due to body fluid circulation, and a high retention rate may not be maintained at the administrated part.
Further, by the method according to Patent Literature 5, a collagen-binding domain is limited to what derived from fibronectin. Although bFGF is disclosed as a bone formation promoting protein, its actual effect is unexplained. Patent Literature 6 is characterized by using a forming particle having a predetermined shape, and despite a description that BMP may be added, an actual evaluation has not been conducted. Even if the component is added, it is presumed that the component will easily leave from the administrated part due to body fluid circulation and is not able to establish a high retention rate. Further, in the case of Patent Literature 7, there is a description that collagen may be mixed as a biocompatible flocculant to formed porous calcium phosphate, however an actual evaluation has not been conducted. Further, since the porous calcium phosphate and the collagen are not fixed together by a covalent bond, the same will easily leave an administrated part due to body fluid circulation, and a sustainable effect is presumed to be hardly attainable. Further, in the case of Patent Literature 8, cross-linked collagen shaped a particulate form is used, however preparation is not easy, and despite a disclosure that a bone growth factor can be added, an actual evaluation has not been conducted. Further, even if a bone growth factor is mixed with the cross-linked collagen, the bone growth factor easily leaves an administration part due to body fluid circulation, and presumably an effect is hardly attainable for a long period.
Regarding artificial joint revision surgery, there are many cases e.g. replacement of a half of femur which can be hardly reconstructed with autologous bone or artificial bone not having an anatomical shape. In such a case there is no other method than transplant of an allogeneic bone maintaining an anatomical shape and having mechanical strengths. Similarly, for a treatment of an intractable fracture, a plate of cortical bone having mechanical strengths is utilized. If a huge allogeneic bone with an anatomical shape is transplanted, it may cause more easily a faulty union or a delayed union at administrated part, compared to a collagen-exposing bone material or crushed bone not having mechanical strengths or an anatomical shape.
In view of the above situation, an object of the present invention is to provide a bone graft material that can maintain the retention rate of a bone growth factor at an administration part, while securing an anatomical shape and mechanical strengths of a bone, and expectedly attain early bone union.
Another object of the present invention is to provide a bone graft material having mechanical strengths and being superior in osteogenic ability, a method for producing a bone graft material, a kit for producing a bone graft material, and a method for forming a bone using the bone graft material.
Solution to Problem
The present inventors have found that a superior osteogenic ability can be expected by binding a fusion protein which a growth factor is bound to a collagen-binding peptide to a bone, that the fusion protein can easily bind to the bone graft substrate exposing at least a collagen fiber by mixing it with the bone graft substrate without a cross-linking reaction or the like, and further that the obtained growth factor anchoring type bone graft material can exert the osteogenic ability at an administratied part for a long time period and consequently early bone union can be expected, thereby established the present invention.
Namely, the present invention provides a growth factor anchoring type bone graft material, wherein a bone graft substrate exposing at least a collagen fiber is bound to a collagen-binding-site-containing growth factor which comprises a growth factor receptor agonist peptide and a collagen-binding peptide (hereinafter also referred to as “CB-GF”).
Further, the present invention provides the growth factor anchoring type bone graft material, wherein the collagen-binding-site-containing growth factor comprises the growth factor receptor agonist peptide, the collagen-binding peptide, and a linker.
Further, the present invention provides the growth factor anchoring type bone graft material, wherein the bone graft substrate is a collagen-exposing bone material or a high-density collagen material.
Further, it provides the growth factor anchoring type bone graft material, wherein the growth factor receptor agonist peptide is a basic fibroblast growth factor.
Further, the present invention provides a method for producing a growth factor anchoring type bone graft material, wherein the bone graft substrate and the CB-GF are mixed.
Further, it provides the method for producing a growth factor anchoring type bone graft material, wherein the bone graft substrate is a collagen-exposing bone material prepared by treating a bone with an acid and removing an inorganic mineral component dissolved by the acid.
Further, the present invention provides a kit for production of a growth factor anchoring type bone graft material, comprising a solution comprising the CB-GF and a bone graft substrate.
Further, the present invention provides a kit for production of a growth factor anchoring type bone graft material, comprising a solution comprising the CB-GF and a collagen-exposing bone material preparation solution.
Further, it provides a method for forming a bone, wherein the growth factor anchoring type bone graft material is transplanted to a bone defect region and/or a non-union region.
Further, the present invention provides the method for forming a bone, wherein the growth factor anchoring type bone graft material is prepared by preparing a collagen-exposing bone material by crushing a bone and treating the same with an acid for 1 to 60 min, and binding the CB-GF to the collagen-exposing bone material.
Advantageous Effects of Invention
A growth factor anchoring type bone graft material of the present invention which a growth factor receptor agonist peptide is bound to a bone graft substrate exposing at least collagen fiber through a collagen-binding peptide of the bone graft substrate, is entirely derived from biogenic substances, and has excellent affinity for an organism and safety.
The growth factor anchoring type bone graft material of to the present invention can be produced easily by simply mixing a bone graft substrate exposing at least a collagen fiber with a CB-GF prepared in advance to be bound together.
Since the growth factor anchoring type bone graft material of the present invention can utilize the bone forming activities of both the bone graft substrate exposing at least a collagen fiber and a growth factor, a good union effect can be exerted even for a case in which union is difficult at the application site of the bone.
Since the kit for production of a growth factor anchoring type bone graft material of the present invention can prepare a collagen-exposing bone material in a short time, it can be used easily at the time of autologous bone grafting.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a diagram showing the results concerning binding capability between a bone graft substrate and an EGF-PKD-CBD fusion protein which is a CB-GF having an EGF as a growth factor receptor agonist peptide; FIG. 1A shows the evaluation results concerning the binding capability between a bone material derived from an epiphysis as a source material prior to a collagen-exposing treatment, and the EGF-PKD-CBD fusion protein; FIG. 1B is a diagram showing the results concerning the binding capability between the bone material after a collagen-exposing treatment and the EGF-PKD-CBD fusion protein;
FIG. 2 is a diagram showing the results concerning binding capability between a bone material using a diaphysis instead of an epiphysis in FIG. 1 and an EGF-PKD-CBD fusion protein, FIG. 2A shows the evaluation results concerning the binding capability between a bone material derived from a diaphysis prior to a collagen-exposing treatment, and an EGF-PKD-CBD fusion protein; FIG. 2B is a diagram showing the results concerning the binding capability between the bone material from a diaphysis after a collagen-exposing treatment and the EGF-PKD-CBD fusion protein;
FIG. 3 is a diagram showing the results concerning binding capability between a bone graft substrate and a bFGF-PKD-CBD fusion protein which is a CB-GF having a bFGF as a growth factor receptor agonist peptide in Example 2; FIG. 3A shows the evaluation results concerning the binding capability between a bone material derived from an epiphysis as a source material prior to a collagen-exposing treatment, and a bFGF-PKD-CBD fusion protein; FIG. 3B is a diagram showing the results concerning the binding capability between the bone material after a collagen-exposing treatment and the bFGF-PKD-CBD fusion protein;
FIG. 4 is a diagram showing the results concerning binding capability between a bone graft substrate using a diaphysis instead of an epiphysis in FIG. 3 and a bFGF-PKD-CBD fusion protein, FIG. 4A shows the evaluation results concerning the binding capability between a bone material derived from a diaphysis prior to a collagen-exposing treatment, and a bFGF-PKD-CBD fusion protein; FIG. 4B is a diagram showing the results concerning the binding capability between the bone material from a diaphysis after a collagen-exposing treatment and the bFGF-PKD-CBD fusion protein;
FIG. 5 is a diagram showing the results of Example 3; FIG. 5A shows the results of a group of bone graft substrates binding a bFGF-PKD-CBD fusion protein; FIG. 5B shows the results of a group of a crushed bone derived from an epiphysis;
FIG. 6 is a diagram showing the area of callus in Example 3;
FIG. 7 is a diagram showing the results of Example 4;
FIG. 8 is a diagram showing the new bone volume in Example 5;
FIG. 9 is a diagram showing the new bone volume in Example 6;
FIG. 10 is a diagram showing the new bone volume in Example 7;
FIG. 11 is a diagram showing a time series change of soft X ray images in Example 8; and
FIG. 12 is a figure illustrating types of bacterial collagenases having a collagen-binding peptide (CBD) and the CBDs.



DESCRIPTION OF EMBODIMENTS
The first aspect of the present invention is a growth factor anchoring type bone graft material characterized in that a bone graft substrate exposing at least a collagen fiber is bound to a CB-GF.
(1) Growth Factor Anchoring Type Bone Graft Material
A bone is constituted with network-formed collagen fibers and hydroxyapatite deposited thereon, and most part of organic substances of a bone is collagen. In a collagen molecule 3 polypeptide chains are bound in a helical fashion, and a large number of the molecules associate in vivo to form insoluble fibers. A collagen exposure-treated matrix (demineralized bone matrix=DBM) prepared by treating a bone with an acidic solution or a chelating reagent to remove nearly completely inorganic substances contains active substances. The substances differentiate undifferentiated mesenchymal cells existing in subcutaneous tissues and muscles to osteoblasts to promote bone formation. The DBM is used as a bone graft material, natural mechanical strengths of a bone, however, have been lost because the same has been demineralized nearly completely. A “growth factor anchoring bone graft material” of the present invention is to use a bone graft substrate exposing at least a collagen fiber. For example, a bone graft substrate which at least a part of inorganic substances is removed from a bone to expose collagen fibers on the bone surface can be used. Such a bone graft substrate to which a CB-GF is bound retains highly its anatomical shape and excels in dynamically, because a large amount of mineral remains in the substrate. In such a bone graft substrate, collagen fibers exist therein without degradation, and the CB-GF can be bound thereto simply by mixing with the bone graft substrate, and therefore production is easy.
The growth factor anchoring type bone graft material of the present invention can be expected synergistic bone forming activity by a growth factor, in addition to the osteogenic ability owned inherently by the bone graft substrate exposing at least a collagen fiber. Furthermore, since the growth factor is bound to the bone graft substrate, it can stay long at a grafted site and promote sustained bone formation. Additionally an autologous bone is used as a source material of the bone graft substrate, it is advantageous in that an immunological rejection reaction can be avoided.
Although there is no particular restriction on the amount of the CB-GF to be bound to the bone graft substrate for the growth factor anchoring bone graft material of the present invention, with respect to 1 mg (dry weight) of a bone graft substrate a CB-GF is bound preferably in an amount of 0.01 to 1 nmol, preferably 0.1 to 1 nmol, and more preferably 0.5 to 1 nmol. Even if the CB-GF is bound beyond 1 nmol, the increasing rate of bone formation is not improved any more; and if it is below 0.01 nmol, the effect of the bound CB-GF may occasionally not be attainable sufficiently.
With respect to a growth factor anchoring bone graft material of the present invention, it is possible that a bone is subjected to a collagen-exposing treatment to prepared the bone graft substrate at the time to use, binding thereto a CB-GF, thereafter it is used as a bone graft material; or alternatively a growth factor anchoring bone graft material prepared in advance by binding a CB-GF to a bone graft substrate and dried for preservation can be used by suspending it in a buffer solution when needed. When a collagen-binding peptide included in the growth factor anchoring bone graft material binds to a collagen fiber by means of its stereostructure, it is preferable to suspend it in a buffer solution that can secure the stereostructure. Examples of such a buffer solution include a phosphate buffer solution of pH 7.4 and a Tris buffer solution.
The growth factor anchoring bone graft material of the present invention can be administered locally for the purpose of increasing bone density, increasing bone mineral density, or increasing new bone similarly to a conventional bone graft material such as an autologous bone graft material. For example, by an administration through a transplant or the like to a bone defect region or a non-union region suffered after tumor curettage or artificial joint revision surgery, bone formation can be promoted. It can be used favorably especially for cases requiring a bone graft material maintaining an anatomical shape and mechanical strengths, such as artificial joint revision surgery, and intractable fracture treatment.
(2) CB-GF
With respect to a CB-GF to be used in the present invention, there is no particular restriction on its structure or production method, insofar as it includes a growth factor receptor agonist peptide (hereinafter also referred to as “GF site”) and a collagen-binding peptide (hereinafter also referred to as “CB site”), and both of the peptides may be bound chemically, or it may be a fusion protein including a GF site and a CB site. In this case, the CB site may be binding directly or through a linker composed of a polypeptide fragment with the GF site. Additionaly, 2 polypeptides of the GF site and the CB site may be cross-linked by a reagent including disuccinimidyl glutarate or glutaraldehyde through an amino group. Further, a polypeptide is derivatized by succinimidyl-4-hydrazinonicotinate acetone hydrazone, and the other polypeptide is derivatized by succinimidyl-4-formyl benzoate, and then two derivatized polypeptides may be mixed for cross-linking through an amino group. According to the present invention, the two may be linked by a crosslinking agent other than polypeptides or other compounds to bind the GF site and the CB site.
(i) Collagen-binding peptide
A “collagen-binding peptide” constituting the CB-GF to be used in the present invention is a functional site to bind a growth factor receptor agonist peptide to the bone graft substrate. Although a growth factor exerts bone forming activity as described above, it cannot be expected sustained bone forming activity because a low local residual ratio by systemic administration such as an intravenous injection. In the present invention, a bone graft substrate exposing at least a collagen fiber is used as a bone graft material, the CB-GF including a GF site and a CB site prepared in advance is mixed with the bone graft substrate to bind a growth factor receptor agonist to the bone graft substrate.
As a method for binding a GF site to a bone graft substrate, a method for binding a bone graft substrate such as a collagen-exposing bone material to a specific component by a chemical cross-linking reaction has been known, for example, as shown in Patent Literature 2. However, by the method, an operation of the reaction is troublesome, and a crosslinking agent may occasionally remain in the collagen-exposing bone material. On the other hand, by the present invention using the CB-GF, the GF site can be bound to the collagen-exposing bone material through a CB site in the CB-GF, without using a crosslinking agent or other chemical components. The growth factor anchoring type bone graft material of the present invention can be prepared easily, and is superior in safety since a crosslinking agent is not used. Further, it is superior in retention of the mechanical strengths and the anatomical shape of the collagen-exposing bone material.
In the present invention, a “CB site” may include widely what can bind at least a part of collagen fibers. Examples of a polypeptide bindable to a collagen fiber include a collagenase-derived collagen binding site. Examples of a structural gene for the collagenase-derived collagen binding site include a DNA fragment including a base sequence of base Nos. 3001 to 3366 of a gene (GenBank Accession Number D29981) of Clostridium histolyticum collagenase (hereinafter occasionally referred to as “ColH”) as set forth in SEQ ID NO: 1. The DNA fragment codes for an amino acid sequence specified by GenBank Accession Number BAA06251. Referring to FIG. 12, a catalytic site represented by CD and a collagen binding site represented by CBD are included and the base sequence of base Nos. 3001 to 3366 corresponds to a CBD. Similarly, Clostridium histolyticum collagenase (hereinafter occasionally referred to as “ColG”) specified by GenBank Accession Number BAA77453, Clostridium limosum collagenase specified by ditto BAC57532, Clostridium septicum collagenase specified by ditto BAC57535, Clostridium perfringens collagenase specified by ditto A36866, Clostridium novyi collagenase specified by ditto BAC57545, Clostridium bifermentans collagenase specified by ditto BAC57541, Clostridium sordellii collagenase specified by ditto BAC57550, Clostridium tetani collagenase specified by ditto AAO37456, Clostridium botulinum collagenase specified by ditto CBO1620, Clostridium sporogenes collagenase specified by ditto BAC57538, Bacillus cereus collagenase specified by ditto NP—833262, Bacillus cereus collagenase specified by ditto NP—979836, Bacillus cereus collagenase specified by ditto NP—833262, Bacillus cereus collagenase specified by ditto NP—979836, Bacillus anthracis collagenase specified by ditto NP—845854, Bacillus thuringiensis collagenase specified by ditto YP—037608, Bacillus cereus collagenase specified by ditto NP—832902, Bacillus anthracis collagenase specified by ditto NP—845590, Bacillus cereus collagenase specified by ditto NP—830373, Bacillus thuringiensis collagenase specified by ditto YP—034814, Bacillus anthracis collagenase specified by ditto NP—843090, Bacillus cereus collagenase specified by ditto NP—976942, and other collagen-binding peptides derived from a bacterial collagenase may be used similarly. Meanwhile, a “CB site” to be used in the present invention is required to bind to a collagen fiber of the bone graft substrate exposing at least a collagen fiber to the extent that the growth factor can be retained there, and therefore it is not necessary to contain the entire amino acid sequence of a collagenase-derived collagen binding site. For example, the collagen-binding peptide having 90% homology with the base sequence constituting a CBD in the amino acid sequence may be favorably used. There is no particular restriction on a binding method, and, for example, it may be bound with an affinity for a part of collagen fibers exposing out of a surface of the collagen-exposing bone material.
(ii) Growth factor receptor agonist peptide
A GF site constituting a CB-GF to be used in the present invention is a site for exerting a function of a growth factor or the like by binding to a bone graft substrate. Examples of a growth factor include an epithelial growth factor (EGF), a fibroblast growth factor (FGF), and a platelet-derived growth factor (PDGF), and a growth factor receptor agonists exerting such actions widely may be used. Further growth factors such as TGF-β, IGF-1, and BMP do not exert a heterotopic bone inducing activity but exert a bone forming activity, they can promote healing of fracture when applied to a fractured part.
As a structural gene for such a growth factor receptor agonist, especially use of a basic fibroblast growth factor is preferable. Examples of such a basic fibroblast growth factor include a DNA fragment composed of a base sequence of base Nos. 468 to 932 of the Homo sapiens fibroblast growth factor 2 (basic) gene (NCBI Reference Sequence Accession Number NM—002006.4) as set forth in SEQ ID NO: 2. As a structural gene for an epithelial growth factor, there is also cDNA (SEQ ID NO: 3) of preproEGF (GenBank Accession Number U04842) of Rattus norvegicus. The amino acid sequence of preproEGF encoded by the DNA is set forth in SEQ ID NO: 4.
As a GF site a basic fibroblast growth factor (bFGF) may be used favorably in the present invention. Since a basic fibroblast growth factor is superior in osteogenic ability, if the CB-GF bound to a basic fibroblast growth factor as a constituent growth factor (hereinafter referred to as “CB-bFGF”) is bound to the bone graft substrate the uniting ability between a recipient bed bone and a grafted bone is superior. A CB-GF bound to an epithelial growth factor (EGF) in place of a basic fibroblast growth factor is referred to as CB-EGF.
(iii) Linker
A CB-GF may be used what is bound to the CB site and the GF site through a linker. By insertion of a linker the CB site and the GF site can be isolated by a predetermined gap width, thus each site can independently fully exert each function. As the result, by insertion of the linker the CB-GF can be bound stronger to collagen fibers than the CB-GF without the linker.
Examples of such a linker include a peptide fragment which does not have a specific three-dimensional structure and is composed of amino acids, such as serine, threonine, proline, asparaginic acid, glutamic acid, and lysine. Further, as such a linker an amino acid sequence derived from the ColH may be used favorably. More specifically, a polycystic kidney disease I domain (hereinafter referred to as “PKD”) of the ColH may be used favorably. Additionally, a PKD derived from another bacterial collagenase may be also used favorably as the linker. This is because the collagen binding ability of the CBD is reinforced by coexistence of the PKD. Such a linker derived a bacterial collagenase is depicted in FIG. 12 as PKD. Incidentally, such a linker should preferably be resistant to a peptide hydrolase or the like contained in a human circulatory liquid, the local residual performance of the GF site is enhanced and bone formation can be persistently promoted.
(3) Bone Graft Substrate
A “bone graft substrate” to be used in the present invention is the bone graft substrate exposing at least a collagen fiber. Examples of the bone graft substrate include a collagen-exposing bone material and a high-density collagen material.
(i) Collagen-exposing bone material
As the collagen-exposing bone material, for example, the collagen-exposing bone material such as crushed bone which is removed at least a part of an inorganic mineral component from the bone may be used favorably. It is not limited to a so-called complete decalcified bone, namely a bone from which all the contained inorganic mineral component is removed. Thereby mechanical strengths of a bone can be secured and the anatomical shape of the same can be retained. By removing a part of the inorganic mineral component, collagen fibers contained in a bone are exposed to a bone surface, and the CB-GF can be bound through the collagen-binding peptide.
A “bone” to be used of the present invention may be any of autologous bone, allogeneic bone, and heterologous bone. Heterologous bone other than human may be from any of primates, such as monkey, baboon, and chimpanzee, swine, cattle, horse, goat, sheep, dog, cat, rabbit, guinea pig, mongolian gerbil, hamster, rat, and mouse. A “collagen-exposing bone material” contains in addition to collagen richly a growth factor, and various peptides and small proteins, maintaining the osteogenic ability. In the present invention, by using a collagen-exposing bone material a growth factor contained in the bone material can be efficiently bound, and the anatomical shape, the mechanical strengths, and the bone inducing potency of a bone can be utilized effectively.
The collagen-exposing bone material to be used in the present invention can be prepared by immersing a bone in an acid solution to expose collagen fibers. Prior to the acid treatment a treatment for removing soft tissues, or a treatment with an organic solvent such as alcohol for removing bone marrow, blood, and lipid, may be conducted.
A bone collected in a block form may be used after shaping into a form corresponding to a bone defect region, or crushing also. When a bone is crushed, the shape may be irregular, and the size may be not uniform. A treatment step for crushing a bone substrate to an appropriate particle size is not limited to before the collagen-exposing treatment, and it may be conducted simultaneously with the collagen-exposing treatment, or conducted after the collagen-exposing treatment. The crushing treatment can be carried out usually with a commonly used a crusher or a mixer, and in either of a wet state and a dry state of a bone substrate. As for the particle size, for example, the largest diameter may be in a range of 50 to 5000 μm, preferably 50 to 1000 μm, and more preferably 50 to 2000 μm.
As for the collagen-exposing bone material to be used in the present invention, a bone which is removed at least a part of an inorganic mineral component so as to expose collagen fibers out of a bone surface may be favorably used. Collagen fibers are required to be exposed from bone tissues to the extent that a CB-GF can bind thereto. A content of calcium can be used as an indicator for removal of an inorganic mineral component. The relative calcium content compared to the value before a collagen-exposing treatment should be reduced up to 95 to 10%, preferably 95 to 40%, more preferably 95 to 60%, and especially preferably 95 to 80%. By mixing a CB-GF thereafter, it can be bound to the collagen-exposing bone material. Conventionally, as a bone graft substrate a complete decalcified bone which a calcium component has been removed to the extent possible is used in general. In the present invention an inorganic mineral component is, however, required to be removed only in the above range, the collagen-exposing treatment time can be shortened.
Such a collagen-exposing treatment on a bone can be performed by dissolving an inorganic mineral component with hydrochloric acid, acetic acid, nitric acid, sulfuric acid, formic acid, or the like. The concentration or treatment conditions may be appropriately selected according to an acid used. For example, in the case 0.6 N hydrochloric acid is used, the temperature is from 0 to 10° C., and the time is from 30 sec to 18 hours, preferably from 60 sec to 6 hours, more preferably from 60 sec to 1 hour, and especially preferably from 60 sec to 2 min. Conventionally, a collagen-exposing treatment was performed by extraction with 0.6 N hydrochloric acid for 3 to 24 hours, the target of the acid extraction was to reduce the calcium content below 5%, as described in Patent Literature 3. However, by the growth factor anchoring type bone graft material of the present invention, it is enough to bind the CB-GF to collagen fibers contained in crushed bone, and further to be killed viable cells to the extent that the antigenecity is removed. By a review of collagen-exposing treatment, it is found that, when a bone is crushed in the largest diameter of 50 to 5000 μm, then treated with 0.6 N hydrochloric acid within the above range, the CB-GF is efficiently bound, the mechanical strengths are kept, and viable cells are killed to reduce antigenicity even if an allogeneic bone is used. The collagen-exposing bone material to be used in the present invention can be used by removing an inorganic mineral component contained in the acid solution after the acid treatment. As a method for removing the inorganic mineral component, the supernatant is removed and washed with water or a phosphate buffer solution, or it may be washed with a chelating reagent.
The collagen-exposing bone material to be used in the present invention may be prepared by using an autologous bone. When allogeneic bone grafting is carried out, the collagen-exposing bone material may be prepared by using a donor bone, according to the above, and preserved in a buffer solution or preserved dry.
(ii) High density collagen material
In the present invention a high-density collagen material may be used as the bone graft substrate. Since a collagen-exposing treatment with an acid for producing a collagen-exposing bone material is not required, the growth factor anchoring type bone graft material can be prepared in a short time.
The density of collagen fibers in the high-density collagen material is from 100 to 800 mg/cm3, preferably from and 300 to 800 mg/cm3, more preferably from 400 to 800 mg/cm3. The mechanical strengths can be superior in the range. The high-density collagen material may be in a sheet form, a columnar form, a spherical form, a polyhedral form, or in another irregular form. Among them the high-density collagen material in a sheet form can be used favorably for e.g. coating a bone surface. There is no particular restriction on a collagen fiber composing the high-density collagen material, and it may be any of collagen types I to XI. Preferably, it is type I. The high-density collagen material is preferably constituted with atelocollagen which a part or all of a telopeptide is removed from a collagen. The high-density collagen material can be prepared by freeze-drying or otherwise drying a solution containing collagen fibers, being pressurizing to the above density and into a sheet form. A commercial product may be also used.
(4) Method for Producing Growth Factor Anchoring Type Bone Graft Material
Since both of the GF site and the CB site constituting the CB-GF to be used in the present invention are peptides, they can be prepared as a fusion protein. When the CB-GF includes a basic fibroblast growth factor (bFGF) as a growth factor receptor agonist, and PKD-CBD derived from ColH as a linker and a CB site, the CB-GF is herein referred to as “bFGF-PKD-CBD”. A method for producing a bFGF-PKD-CBD is disclosed in Non Patent Literature 1, the bFGF-PKD-CBD can be produced by the method. By using a basic fibroblast growth factor (bFGF) as a GF site, and a CBD derived from ColG as a CB site, a bFGF-CBD can be also produced by fusing the two. By using a gene sequence for an epithelial cell growth factor (EGF) instead of a gene sequence for a bFGF, a CB-EGF can be produced similarly as above. Further by using a gene sequence coding for another growth factor receptor agonist, a CB-GF which the growth factor receptor agonist binds to the CB can be produced. As described above, the CB site and the GF site may be cross-linked by a crosslinking agent.
In the present invention the growth factor anchoring type bone graft material maybe produced by mixing the EGF-PKD-CBD, or other CB-GF with the above bone graft substrate. Generally, by adding predetermined amounts of the bone graft substrate and the CB-GF into a phosphate buffer solution, stirring the mixture for 60 sec to 60 min, preferably 5 to 30 min, and more preferably 15 to 30 min at a temperature of 0 to 10° C., or leaving it standing, the CB-GF can be bound to the bone graft substrate.
The growth factor anchoring type bone graft material of the present invention can be easily prepared and used provided that the bone graft substrate is prepared at a conventional autologous bone grafting, then the CB-GF prepared in advance is added immediately the substrate to prepare the growth factor anchoring type bone graft material. In the case of allogeneic bone grafting, the bone graft substrate which is prepared by the above method in advance or preserved in a buffer solution may be used. Furthermore a growth factor anchoring type bone graft material which is prepared by immersing a dried bone graft substrate in a buffer solution and adding the CB-GF thereto may be used as a grafting bone material.
(5) Kit for Production of a Growth Factor Anchoring Type Bone Graft Material
As a kit for production of a growth factor anchoring type bone graft material of the present invention, there are a kit (I) composed of a CB-GF solution and the bone graft substrate, and a kit (II) composed of a CB-GF solution and a collagen-exposing bone material preparation solution.
(i) Kit (I)
A kit (I) is composed of a CB-GF solution and the bone graft substrate. Examples of a bone graft substrate include a donor bone which is removed at least a part of an inorganic mineral component to expose collagen fibers and then preserved in a buffer solution, the same preserved in a dry state, and the high-density collagen material.
The CB-GF solution in the kit (I) is a solution dissolving the CB-GF in a buffer solution in a range of 0.5 to 2.0 mg/mL. Examples of a buffer solution include a phosphate buffer solution of pH 7.0 to 8.0, Tris buffer solution, and a physiological saline solution. Since the bone graft substrate is included in the kit, the growth factor anchoring type bone graft material can be easily prepared by adding the CB-GF solution to the bone graft substrate before transplanting.
(ii) Kit (II)
A kit (II) is composed of a collagen-exposing bone material preparation solution in place of a bone graft substrate, and a CB-GF solution. For example, at an autologous bone grafting, the collagen-exposing bone material can be easily prepared by immersing an autologous bone in the collagen-exposing bone material preparation solution followed by washing. By adding the CB-GF solution to the obtained collagen-exposing bone material followed by mixing, the growth factor anchoring type bone graft material can be prepared. An acid solution such as 0.6 N hydrochloric acid solution, and acetic acid, as well as an acid solution to which a chelating reagent is added, may be used as a collagen-exposing bone material preparation solution. A kit (II) may be used favorably for conducting an autologous bone grafting.
(6) Method for Forming Bone
The growth factor anchoring type bone graft material of the present invention is a bone graft material which the CB-GF including the GF site such as FGF, TGF-β, IGF-1, and PDGF, and the CB site is bound to the bone graft substrate. The osteogenic ability based on the bone graft substrate and the osteogenic effect based on the growth factor can be expected. For a treatment of a bone defect region suffered after tumor curettage or artificial joint revision surgery or a treatment of a non-union (pseudoarthrosis), crushed autologous bone as a graft bone or crushed allogeneic bone as a graft bone has been heretofore used. By using the growth factor anchoring type bone graft material instead of a conventional graft bone, a growth factor can stay for a long period at the grafted site and promote bone formation persistently, thereby forming a bone earlier than in the past.
Specifically, by transplanting the growth factor anchoring type bone graft material to a bone defect region or a non-union region suffered after tumor curettage or artificial joint revision surgery, bone formation can be promoted.
For example, at the time of an autologous bone grafting operation, a graft bone is obtained, crushed in the range of the largest diameter 50 to 5000 μm, and stirred in 0.6 N hydrochloric acid for 1 min to perform a collagen-exposing treatment. Then the obtained collagen-exposing bone material is washed with water, rinsed with a phosphate buffer solution (pH 7.0 to 8.0), added the CB-GF thereto and mixed for approx. 1 to 30 min, thereby preparing a growth factor anchoring type autologous bone graft material. By grafting the same to a bone defect region or a non-union region suffered after tumor curettage or artificial joint revision surgery, an autologous bone grafting can be carried out. Contrary to a conventional autologous bone, the growth factor anchoring type bone graft material of the present invention includes the CB-GF. Therefore excellent bone formation based on the CB-GF can be expected. On an occasion of a fracture or the like early ambulation owing to premature fusion at an affected part becomes possible, so that rehabilitation can be started early. In the case of an allogeneic bone grafting, it is possible to prepare a growth factor anchoring type allogeneic bone graft material before the surgery. Therefore, an allogeneic bone grafting can be carried out effectively within a short operation time and with minimal invasion.
A collagen-exposing bone material preparation solution in the kit (II) can be used for the preparation of the collagen-exposing bone material, and a CB-GF solution in the kit (II) may be used as the CB-GF.
EXAMPLES
Next, the present invention will be specifically described below referring to Examples, provided that the present invention be not restricted in any way by the Examples.
Production Example 1
Production of EGF-PKD-CBD Fusion Protein
(1) A region of base Nos. 3001 to 3366 in DNA (SEQ ID NO: 1) of ColH is a gene fragment coding for a collagen binding domain (CBD). A region of base Nos. 2719 to 3000 in the DNA (SEQ ID NO: 1) is a gene fragment coding for a PKD domain (PKD) of a bacterial collagenase, and can be used for a linker. Therefore, a region of base Nos. 2719 to 3391 in the DNA (SEQ ID NO: 1) including the sites was cut off and inserted it into a SmaI site in a pGEX-4T-2 plasmid in the usual manner.
(2) A DNA (SEQ ID NO: 5) consisting of a base sequence of base Nos. 3308 to 3448 in cDNA SEQ ID NO: 3 of preproEGF of Rattus norvegicus (GenBank Accession Number U04842) was amplified by a PCR method so as to have a BamHI site at the 5′end and one nucleotide (G residue) for alignment of a reading frame of a fusion protein and an EcoRI site at the 3′end. The fragment was inserted into the BamHI-EcoRI site of the expression vector according to the item (1) by an usual manner. The obtained expression plasmid possesses a reading frame (SEQ ID NO: 7) coding for a GST-EGF-PKD-CBD fusion protein (SEQ ID NO: 6).
(3) The obtained expression plasmid (2) above was introduced in Escherichia coli (BL21 Codon Plus RIL) by an electroporation method.
The Escherichia coli was precultured overnight in 50 mL of a 2×YT-G culture medium containing 50 μg/mL of ampicillin and 30 μg/mL of chloramphenicol. To 500 mL of the culture medium 10 mL of the obtained precultured liquid was added and shake-cultured at 37° C. until the turbidity (O. D. 600) of the bacterial suspension became approx. 0.7. To the obtained bacterial suspension, 5 mL of a 0.1 M-aqueous solution of isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and cultured at 37° C. for 2 hours. Then, 5 mL of phenylmethylsulfonyl fluoride (PMSF) solution containing 0.1 M isopropanol was added, and the culture solution was centrifuged at 6,000×g, and 4° C. for 10 min to collect a transformant. Bacterial cells were suspended in 7.5 mL of a phosphate buffered physiological saline solution (PBS) containing 1 mM PMSF, and the cells were destructed by a French press. A 20%-Triton X-100 solution equivalent to 1/19 volume of the suspension was added and stirred at 4° C. for 30 min. The lysate was centrifuged at 15,000×g, and 4° C. for 30 min to obtain a supernatant, and the resulting supernatant was then centrifuged again under the same condition. The supernatant was defined as a cleared lysate solution. To glutathione-sepharose beads (2 mL), the cleared lysate solution was added and stirred at 4° C. for 1 hour to bind a GST-EGF-PKD-CBD fusion protein to the beads. After washing the beads with 12 mL of PBS five times, the beads were suspended in a small amount of PBS and loaded onto a column. The fusion protein was eluted with 50 mM Tris-HCl (pH 8.0) and 10 mM glutathione solution. Five units of thrombin per mg of the fusion protein were added and the mixture was subjected to a reaction at 25° C. for 10 hours to cleave a GST tag. After that, dialysis against 300 mL of PBS at 4° C. for 12 hours was repeated four times. The dialyzed cleavage product was added to a column filled with fresh glutathione-sepharose beads (2 mL) washed with PBS and directly eluted. As a result, the GST tag was removed and EGF-PKD-CBD fusion protein (SEQ ID NO: 6; 225 to 491) without the GST tag was obtained.
Production Example 2
Production of bFGF-PKD-CBD Fusion Protein
Firstly, a DNA fragment (PKD-CBD gene) including a base sequence of base Nos. 2719 to 3391 of the ColH gene set forth in SEQ ID NO: 1 was inserted in an SmaI site of a pGEX-4T-2 plasmid (by GE Healthcare, Japan) in the usual manner. Meanwhile, a DNA fragment (bFGF gene) consisting of a base sequence of base Nos. 468 to 932 in the Homo sapiens fibroblast growth factor 2 (basic) gene (NCBI Reference Sequence Accession Number NM—002006.4) set forth in SEQ ID NO: 2 was amplified by a PCR method so as to have a BamHI site at the 5′end and one nucleotide (G residue) and an EcoRI site at the 3′end. The amplified DNA fragment (bFGF gene) was inserted into the BamHI-EcoRI site plasmid inserted the DNA fragment (PKD-CBD gene) in the usual manner, thereby preparing an expression plasmid. The obtained expression plasmid possesses a reading frame (SEQ ID NO: 9) coding GST-bFGF-PKD-CBD fusion protein (SEQ ID NO: 8). The amino acid sequence of the bFGF-PKD-CBD fusion protein is set forth in SEQ ID NO: 10, and the base sequence coding for the bFGF-PKD-CBD fusion protein is set forth in SEQ ID NO: 11. In the amino acid sequence according to SEQ ID NO: 10, the N-terminal 2 amino acid residues Gly-Ser are a part of a recognition site of a GST tag cleavage enzyme (thrombin protease). The expression plasmid was introduced in Escherichia coli (BL21 Codon Plus RIL, by Stratagene) by an electroporation method to produce a transformant.
The transformant was precultured overnight in 50 mL of a 2×YT-G culture medium containing 50 μg/mL of ampicillin and 30 μg/mL of chloramphenicol. Ten mL of the obtained preculture solution was added to 500 mL of the culture medium and was shake-cultured at 37° C. until the turbidity (O. D. 600) of the bacterial suspension reached approx. 0.7. To the obtained bacterial suspension 5 mL of a 0.1 M isopropyl-β-D-thiogalactopyranoside (IPTG) aqueous solution was added and the mixture was cultured at 37° C. for 2 hours. After adding 5 mL of an isopropanol solution containing 0.1 M phenylmethylsulfonyl fluoride (PMSF), the bacterial suspension was centrifuged at 6000×g and 4° C. for 10 min to collect the transformant. The transformant was suspended in 7.5 mL of 50 mM Tris-HCl (pH 7.5), 0.5M NaCl and 1 mM PMSF, and the cells were destructed by a French press. To 19 volume of the suspension, 1 volume of a 20% Triton (registered trademark) X-100 was added and stirred at 4° C. for 30 min. The obtained bacterial suspension was centrifuged at 15,000×g and 4° C. for 30 min and the supernatant was recovered. The obtained supernatant was further centrifuged at 15,000×g and 4° C. for 30 min and the supernatant was recovered. The supernatant was defined as a clarified lysate. The clarified lysate was added to 2 mL of glutathione-sepharose beads and stirred at 4° C. for 1 hour. After washing the beads 5 times with 12 mL of 50 mM Tris-HCl (pH 7.5) and 0.5M NaCl, the beads were suspended in small amount of 50 mM Tris-HCl (pH 7.5) and 0.5M NaCl, and filled in a column. Then the GST-bFGF-PKD-CBD fusion protein was eluted therefrom with an elution liquid (50 mM Tris-HCl (pH 8.0), 0.5M NaCl and 10 mM glutathione). Thrombin in an amount of 5 units per 1 mg of the fusion protein was added and allowed to react at 25° C. for 10 hours. The obtained reaction solution was added to 1 mL of heparin-sepharose beads and stirred at 4° C. for 3 hours allowing the bFGF-PKD-CBD fusion protein to bind to the beads. After discarding the supernatant gently, the beads were washed 3 times with 12 mL of 50 mM Tris-HCl (pH 7.5) with 0.5 M NaCl. The beads were filled in a column and the protein was eluted with 10 mL of 50 mM Tris-HCl (pH 7.5) with the salt gradient of NaCl from 0.5 to 2M, to obtain the bFGF-PKD-CBD fusion protein (SEQ ID NO: 10).
Production Example 3
Production of bFGF-CBD Fusion Protein
A DNA fragment including a base sequence of base Nos. 4011 to 4358 of the ColG gene set forth in SEQ ID NO: 12 was amplified by a PCR method so as to have an SmaI site at the 5′end, and an XhoI site at the 3′end. The fragment was inserted between an SmaI site and an XhoI site of a pGEX-4T-2 plasmid in the usual manner. Meanwhile, a DNA fragment (bFGF gene) consisting of a base sequence of base Nos. 468 to 932 of the Homo sapiens fibroblast growth factor 2 (basic) gene (NCBI Reference Sequence Accession Number NM—002006.4) set forth in SEQ ID NO: 2 was amplified by a PCR method so as to have a BglII site at the 5′end, and a nucleotide (base G) and an EcoRI site at the 3′end. The amplified DNA fragment (bFGF gene) was inserted in the usual manner in a BamHI-EcoRI site of the plasmid into which the DNA fragment (CBD gene) was inserted to prepare an expression plasmid. The expression plasmid possesses a reading frame coding for the GST-bFGF-CBD fusion protein (SEQ ID NO: 13). The amino acid sequence of the bFGF-CBD fusion protein is an amino acid sequence corresponding to base Nos. 720 to 1503 of the base sequence set forth in SEQ ID NO: 13. In the amino acid sequence, the N-terminal 2 amino acid residues Gly-Ser are a part of a recognition site of a GST tag cleavage enzyme (thrombin protease). The expression plasmid was introduced in Escherichia coli (BL21 Codon Plus RIL, by Stratagene) by an electroporation method to produce a transformant.
A bFGF-CBD fusion protein was produced identically with the production example 2, except that this transformant was used.
Example 1
A femur was obtained from a 2 months old male Wistar rat and subjected to defatting freeze-drying.
The bone tissue was divided to epiphysis and diaphysis, and each of them was crushed to an average particle size of 50 to 300 μm. To 40 mg of each crushed bone 1 mL of 0.6 N hydrochloric acid was added and the mixture was stirred at a temperature of 4° C. for 18 hours. Then the mixture was washed twice with a pH 7.4-phosphate buffer solution to prepare a collagen-exposing bone material of epiphysis or diaphysis.
To the crushed bone (bone material before collagen-exposing treatment) of epiphysis 5 mg, 10 mg, 20 mg, 40 mg, 80 mg, and 160 mg, 0.2 mL each of a phosphate buffer solution and 1.16 nmol of the EGF-PKD-CBD fusion protein obtained in the production example 1 were added and mixed for 30 min. After mixing, a supernatant was collected and the amount of the fusion protein contained in the supernatant was examined by SDS-PAGE. The results are shown in FIG. 1A. In FIG. 1A are shown from left molecular weight marker (Marker), stock solution of the EGF-PKD-CBD fusion protein obtained in the production example 2 (con), collagen (CP) 5 mg, crushed bone (BP) 5 mg, crushed bone (BP) 10 mg, crushed bone (BP) 20 mg, crushed bone (BP) 40 mg, crushed bone (BP) 80 mg, and crushed bone (BP) 160 mg.
While to each of 5 mg, 10 mg, 20 mg, 40 mg, 80 mg and 160 mg (weight before collagen-exposing treatment) of the collagen-exposing bone material (DBP) from epiphysis, instead of the crushed bone of epiphysis (bone material before collagen-exposing treatment), 0.2 mL of a phosphate buffer solution and 1.16 nmol of the EGF-PKD-CBD fusion protein obtained in the production example 1 were added and mixed for 30 min. After mixing, a supernatant was collected and the amount of the fusion protein contained in the supernatant was examined by SDS-PAGE. For comparison instead of the EGF-PKD-CBD fusion protein 1.16 nmol of bovine albumin was added and the same procedures were carried out. The results are shown in FIG. 1B. In FIG. 1 to FIG. 4, groups using the crushed bone are referred to as Pre-decalcification (BP) and groups using the collagen-exposing bone material are referred to as Post-decalcification (DBP).
Further, using a crushed bone of diaphysis instead of the crushed bone of epiphysis, and using a collagen-exposing bone material of diaphysis instead of the collagen-exposing bone material of epiphysis, the same procedures were carried out, and the binding activities of the EGF-PKD-CBD fusion protein were evaluated. The results are shown in FIG. 2A and FIG. 2B respectively.
Comparing FIG. 1A and FIG. 1B, in FIG. 1A the amount of the fusion protein in the supernatant is constant irrespective of the amount of the crushed bone, in FIG. 1B the amount of the fusion protein in the supernatant is decreased in proportion to increase in the amount of the collagen-exposing bone material. Since the EGF-PKD-CBD fusion protein not bound to the collagen-exposing bone material is present in the supernatant, it is presumed that as the amount of collagen-exposing bone material was increased, more EGF-PKD-CBD fusion protein was bound to the collagen-exposing bone material. Meanwhile, in the case of epiphysis, even with respect to bovine albumin the residual amount in a supernatant is decreased depending on the amount of the collagen-exposing bone material similarly to the EGF-PKD-CBD fusion protein, to indicate that the binding capability of a protein is increased by the collagen-exposing treatment.
Further, comparing FIG. 1B and FIG. 2B with respect to the binding amount of the EGF-PKD-CBD fusion protein to the collagen-exposing bone material, the binding amounts to the collagen-exposing bone material derived from epiphysis and to the collagen-exposing bone material derived from diaphysis were nearly the same. On the other hand, as obvious from the comparison of FIG. 1B and FIG. 2B, the amount of BSA in the supernatant was larger for diaphysis. This means that the binding amount of albumin depends on a bone part. It is presumed that of the present invention, the EGF-PKD-CBD fusion protein could be anchored to a crushed bone irrespective of a used bone part.
Example 2
The same procedures were carried out as in Example 1, except that the bFGF-PKD-CBD fusion protein obtained in the production example 2 was used instead of the EGF-PKD-CBD fusion protein, and the binding activities of the bFGF-PKD-CBD fusion protein to the crushed bone and the collagen-exposing bone material derived from epiphysis, and the crushed bone and the collagen-exposing bone material derived from diaphysis respectively were examined. The results of the binding activities of the bFGF-PKD-CBD fusion protein to the crushed bone and the collagen-exposing bone material derived from epiphysis are shown in FIG. 3A and FIG. 3B, and the results of the binding activities of the bFGF-PKD-CBD fusion protein to the crushed bone and the collagen-exposing bone material derived from diaphysis are shown in FIG. 4A and FIG. 4B.
Comparing FIG. 3A and FIG. 3B, the amounts of the fusion protein in the supernatant were decreased with increase in the amount of the crushed bone and also of the collagen-exposing bone material. However, for the collagen-exposing bone material the dependence on the amount of bone was higher than for the crushed bone to indicate that the binding capacity of the bFGF-PKD-CBD fusion protein was improved by a collagen-exposing treatment.
Further, by comparing FIG. 3 and FIG. 4, with respect to the collagen-exposing bone material derived from diaphysis by addition of 80 mg, the bFGF-PKD-CBD fusion protein in the supernatant was nearly disappeared, while with respect to the collagen-exposing bone material derived from epiphysis by addition of 40 mg the same in the supernatant was nearly disappeared, to indicate that the binding capability of the bFGF-PKD-CBD fusion protein was higher for a collagen-exposing bone material derived from epiphysis than for a collagen-exposing bone material derived from diaphysis. It was also indicated that of the present invention a CB-GF can be anchored to the collagen-exposing bone material irrespective of a used bone part and a used CB-GF type.
Example 3
Six 2 months old male Wistar rats were divided to 2 groups of 3 each. Both of the groups were anesthetized with Nembutal on the anterior femoral, and a collagen-exposing bone material (growth factor anchoring type bone graft material), in which 20 mg of the bFGF-PKD-CBD fusion protein bind prepared in the production example 2 was bound to 20 mg (weight before collagen-exposing treatment) of a collagen-exposing bone material prepared identically with Example 1 was transplanted on the anterior femoral periosteum of one group, and 20 mg of a crushed bone of epiphysis prepared in Example 1 was transplanted on the anterior femoral periosteum of the other group.
Bone formation was observed with time by taking a soft X ray photograph every week. The results of the transplant of the collagen-exposing bone material with the bound bFGF-PKD-CBD fusion protein are shown in FIG. 5A, and the results of the transplant of the crushed bone of epiphysis are shown in FIG. 5B.
As shown in FIG. 5A, when a growth factor anchoring type bone graft material was transplanted on the anterior femoral periosteum, after approx. 1 week from the transplant a bone tissue was observed (arrow) in the vicinity of the growth factor anchoring type bone graft material, and after approx. 2 weeks a bone tissue with certain thickness was observed in a wider range. On the contrary, in the control group transplanted with a crushed bone, even 2 weeks after the transplant, no bone tissue could be observed in the vicinity of the crushed bone. Meanwhile, the area of a new bone tissue (callus) is shown in FIG. 6. The black bar is for the control group, and the white bar is for the group bound to the bFGF-PKD-CBD fusion protein.
It has become clear that a growth factor anchoring type bone graft material of the present invention can form a bone tissue faster than a conventional allogeneic bone grafting.
Example 4
From a 2 months old male Wistar rat a femur was obtained and subjected to defatting freeze-drying.
The diaphysis of the bone tissue was crushed to an average particle size of 50 to 300 μm. The crushed bone was divided to 3 groups of 40 mg each (weight before collagen-exposing treatment), and the group 1 was for a not collagen exposure-treated crushed bone (BP), and group 2 and group 3 were for a collagen exposure-treated crushed bone (DBP). To the collagen exposure-treated crushed bone (DBP) groups, 1 mL of 0.6 N hydrochloric acid was added and stirred at a temperature of 4° C. for 1 min or 18 hours. The mixture was then washed twice with a pH 7.4 phosphate buffer solution and used as a bone graft substrate of diaphysis.
Next, to each of 40 mg of the crushed bone (BP) of diaphysis, the group of the 1 min-collagen-exposing treatment, and the group of the 18 hour-collagen-exposing treatment, 0.2 mL of a phosphate buffer solution and 1.16 nmol of the bFGF-PKD-CBD fusion protein obtained in the production example 2 were added and blended for 30 min. After the blending a supernatant was collected and the amount of the fusion protein in the supernatant was examined by SDS-PAGE. The results are shown in FIG. 7. The calcium content of the group of the 1 min-collagen-exposing treatment was 90 mass-%, and the calcium content of the group of the 18 hour-collagen-exposing treatment was 10 mass-%.
In FIG. 7 are shown from left molecular weight marker (Marker), stock solution (con), crushed bone (BP), crushed bone with 1 min-collagen-exposing treatment (DBP), and crushed bonewith 18 hour-collagen-exposing treatment (DBP).
As shown in FIG. 7, for the crushed bone (BP) a fusion protein is observed in the supernatant, on the contrary for both of the crushed bonewith 1 min-collagen-exposing treatment (DBP), and crushed bonewith 18 hour-collagen-exposing treatment (DBP), no fusion protein is observed in supernatants to indicate that a CB-GF can be bound to the bone graft substrate even after a short time collagen-exposing treatment.
Example 5
Sixty four 10 weeks old male Wistar rats were divided to 4 groups of 16 each. A growth factor anchoring type bone graft material was prepared by reacting 20 mg (weight before collagen-exposing treatment) of a demineralize bone material of diaphysis prepared as in Example 1, with 1.16 nmol of a bFGF, 0.29 nmol of a bFGF-PKD-CBD fusion protein, or 1.16 nmol of a bFGF-PKD-CBD fusion protein, and transplanted on the anterior periosteum of the femoral diaphysis.
After 1 week and 2 weeks from the transplant, the femora of 8 rats of each group were obtained and the new bone volume was measured using a micro-CT. Meanwhile, a phosphate buffer solution (PBS) and the collagen-exposing bone material were reacted and transplanted as the control. The results are shown on FIG. 8.
The white bar is for the control group, the grey bar is for the 1.16 nmol bFGF group, the black bar is for the 0.29 nmol bFGF-PKD-CBD fusion protein group, and the gradation column is for the 1.16 nmol bFGF-PKD-CBD fusion protein group. The “a” means significant difference to the control group, and the “b” means significant difference to the 1.16 nmol bFGF group.
FIG. 8 shows that the new bone amount of the 1.16 nmol bFGF-PKD-CBD fusion protein group after 1 week was significantly larger than the 1.16 nmol bFGF group. After 2 weeks, the amounts of a new bone of both the 0.29 nmol bFGF-PKD-CBD fusion protein group and the 1.16 nmol bFGF-PKD-CBD fusion protein group were significantly larger than the bFGF group. It has been shown that by using the collagen-exposing bone material and the bFGF-PKD-CBD fusion protein according to the present invention, bone formation can be promoted at a low dose for a long term.
Example 6
Thirty-two 10 week-old male Wistar rats were divided to 2 groups of 16 rats each. After reacting 20 mg (weight before collagen-exposing treatment) of the collagen-exposing bone material of diaphysis prepared identically with Example 1, with the bFGF-PKD-CBD, or the bFGF-CBD fusion protein obtained in the production example 3, the product was transplanted on the anterior periosteum of femoral diaphysis. The reaction amount was 0.58 nmol for both the groups.
After 1 week and 2 weeks from the transplant, the femora of 8 rats of each group were obtained and the new bone volume was measured using a micro-CT. The results are shown in FIG. 9. The new bone amount after 2 weeks from the transplant tends to be large in the bFGF-CBD fusion protein group. It has been shown that by changing the collagen binding domain the controlled release period or the bone formation amount can be controlled according to the present invention.
Example 7
Eighty 10 week-old male Wistar rats were divided to 4 groups of 20 rats each. A bone graft material formed by reacting a sheet-formed high-density collagen material (collagen fiber density of 640 mg/cm3, 5 mm×5 mm×100 μm), with 0.58 nmol of bFGF, 0.58 nmol of bFGF-CBD fusion protein, or 0.58 nmol of bFGF-PKD-CBD fusion protein respectively was transplanted on the anterior periosteum of femoral diaphysis. A group transplanted with a reaction product of a phosphate buffer solution (PBS) and the high-density collagen material was defined as the control.
After 1 week and 2 weeks from the transplant, the femora of 10 rats of each group were obtained and the new bone volume was measured using a micro-CT. The results are shown in FIG. 10. The amount of new bone after 1 week from the transplant were the same for the bFGF group, the bFGF-CBD (I) fusion protein, and the bFGF-PKD-CBD (II) fusion protein group, however after 2 weeks the same was significantly high for the bFGF-PKD-CBD (II) fusion protein. According to the present invention, it has been shown that by using the high-density collagen material having high strengths, a graft bone substitute material that can promote bone formation for a long time period can be provided.
Example 8
Six 10 week-old male C57BL/6J mice were divided to 2 groups. To simulate reconstruction of a wide range bone defect suffered after tumor curettage or injury, a 5 mm-bone defect was prepared at the murine femur diaphysis and then a bone was grafted thereto. After bone grafting a bone graft material obtained by reacting the bFGF-PKD-CBD fusion protein prepared as in Example 7 with a sheet-formed high-density collagen material (collagen fiber density of 640 mg/cm3, 5 mm×5 mm×100 μm), was coated thereon. Meanwhile, a group coated with a reaction product of a phosphate buffer solution (PBS) and a sheet-formed high-density collagen material was defined as the control.
The results of temporal change of a mouse of each group are shown in FIG. 11. After 3 weeks from the grafting, vigorous new bone formation is recognizable around the grafted bone in the group coated with a bone graft material, and further that union of the grafted bone and a recipient bed bone was recognized. The above has demonstrated that the bone graft material is useful as a substitute material for an allogeneic cortical bone plate requiring high mechanical strengths.
The present invention is based on Japanese Patent Application No. 2011-108650 filed on 13 May 2011. The description, claims, and drawings of Japanese Patent Application No. 2011-108650 are incorporated herein by reference in its entirety.
INDUSTRIAL APPLICABILITY
A growth factor anchoring type bone graft material of the present invention can be produced easily, and used similarly as a conventional bone graft material. Further, since a growth factor is added, the same is superior in uniting ability of a grafted bone with a recipient bed bone, and therefore useful.

## Claims

1. A growth factor anchoring type bone graft material, wherein a bone graft substrate exposing at least a collagen fiber is bound to a collagen-binding-site-containing growth factor which comprises a growth factor receptor agonist peptide and a collagen-binding peptide,
wherein the bone graft substrate is a high-density collagen material in a sheet form with a collagen fiber density of 100 to 800 mg/cm3, and the collagen-binding-site-containing growth factor is formed by ligating a basic fibroblast growth factor and the collagen-binding peptide through a polycystic kidney disease I domain of a collagenase.

2. A kit for production of a growth factor anchoring type bone graft material wherein a bone graft substrate exposing at least a collagen fiber is bound to a collagen-binding-site-containing growth factor which comprises a growth factor receptor agonist peptide and a collagen-binding peptide,
wherein the bone graft substrate is a high-density collagen material in a sheet form with a collagen fiber density of 100 to 800 mg/cm3, and the collagen-binding-site-containing growth factor is formed by ligating a basic fibroblast growth factor and the collagen-binding peptide through a polycystic kidney disease I domain of a collagenase, which kit comprises: a solution comprising a collagen-binding-site-containing growth factor formed by ligating a basic fibroblast growth factor and the collagen-binding peptide through a polycystic kidney disease I domain of a collagenase;
and a bone graft substrate which is said high-density collagen material in a sheet form with the collagen fiber density of 100 to 800 mg/cm3.