JP3616082B2 - Osteogenic treatment device - Google Patents

Description

【０１１２】
表１に示すように、実施例の骨形成治療デバイス（本発明の骨形成治療デバイス）の骨形成率は、比較例１〜比較例３の骨形成治療デバイスの骨形成率と比較して、３週間後および６週間後では同等もしくは低いものもあったが、９週間後には、いずれのものよりも顕著に高くなった。
【０１１３】
このように、組換えプラスミドと塩基性線維芽細胞増殖因子（ｂＦＧＦ）とを併用することにより、極めて優れた骨形成能を有する骨形成治療デバイスが得られることが明らかとなった。
【０１１４】
また、基体として、ハイドロキシアパタイト多孔質焼結体に代わり、リン酸三カルシウム多孔質焼結体を用いて骨形成治療デバイスを作製して、前記と同様の評価実験を行った結果、前記とほぼ同様の評価結果が得られた。
【０１１５】
また、ＢＭＰをコードする塩基配列として、ヒトＢＭＰ−２をコードする塩基配列に代わり、ヒトＢＭＰ−１、ヒトＢＭＰ−２、ヒトＢＭＰ−３、ヒトＢＭＰ−４、ヒトＢＭＰ−５、ヒトＢＭＰ−６、ヒトＢＭＰ−７、ヒトＢＭＰ−８、ヒトＢＭＰ−９またはヒトＢＭＰ−１２をコードする塩基配列、あるいは、これらを任意に組み合わせて骨形成治療デバイスを作製して、前記と同様の評価実験を行った結果、前記実施例とほぼ同様の評価結果が得られた。
【０１１６】
また、血管形成誘導因子として、塩基性線維芽細胞増殖因子（ｂＦＧＦ）に代わり、血管内皮増殖因子（ＶＥＧＦ）または肝細胞増殖因子（ＨＧＦ）、あるいは、これらを任意に組み合わせて骨形成治療デバイスを作製して、前記と同様の評価実験を行った結果、前記実施例とほぼ同様の評価結果が得られた。
【０１１７】
【発明の効果】
以上述べたように、本発明によれば、極めて迅速な骨形成を可能とし、早期の骨形成治療に貢献することができる。
【０１１８】
特に、本発明では、血管形成誘導因子を併用することにより、骨芽細胞の周囲に新生血管が活発に形成され、骨芽細胞が効率よく増殖して、その結果、迅速な骨形成がなされる。
【０１１９】
このため、各種骨形成治療に際し、遊離骨移植を行う必要がなくなり、採骨部が不要となることから、より安全かつ確実で、合理的な手術が可能となる。
【０１２０】
このようなことから、手術時間や入院期間の短縮を図ることができ、トータルの医療コストの削減、治療クオリティーの向上、患者のＱＯＬの向上を図ることができる。
【０１２１】
また、核酸を保持するベクターを併用することにより、未分化間葉系細胞、炎症細胞、線維芽細胞のような骨形成に関与する細胞内への核酸の取り込みの効率が向上して、結果として、より迅速な骨形成がなされ、前記効果がより向上する。
【０１２２】
また、基体としてブロック体を用いることにより、基体の形状に沿って骨形成が良好に進行するので、移植部位が比較的大きな骨欠損部等である場合に有効である。
【０１２３】
また、基体として、遺伝子治療に特化した多孔質体を用いることにより、核酸および血管形成誘導因子を、より容易かつ確実に基体に担持させることができるとともに、各種の骨形成に関与する細胞や血管形成に関与する細胞が基体内に侵入し易くなり、骨形成にとって有利である。
【０１２４】
また、本発明の骨形成治療デバイスは、保存、取扱いや手術時の加工等が容易である。
【図面の簡単な説明】
【図１】組換えプラスミドの一例を示す遺伝子地図である。[0001]
[Technical field to which the invention belongs]
The present invention relates to an osteogenic treatment device.
[0002]
[Prior art]
In the medical field, research on so-called artificial bones, artificial skin, and artificial organs has attracted attention, and new challenges have already been made from various standpoints in clinical practice. Has also shown great progress.
[0003]
In particular, allotransplantation has problems such as unknown infectious diseases, in addition to the chronic persistence of demand far exceeding supply.
[0004]
In order to avoid these problems, the development of artificially manufactured artificial tissues / artificial organs has been awaited as an alternative to such transplantation of allogeneic tissues. The same applies to bone grafting.
[0005]
Bone formation and repair processes have been elucidated by many researchers, and in addition to the discovery, identification, and separation of osteoinductive factors that are key to their control, it is possible to generate osteoinductive factors using genetic engineering techniques, The knowledge about the mechanism of action is also accumulated.
[0006]
As this osteoinductive factor, bone morphogenic protein (BMP) belonging to the super family of Transforming Growth Factor b (TGFb) has attracted the attention of many researchers. This BMP is an active protein that acts on undifferentiated mesenchymal cells in subcutaneous tissue or muscle tissue to differentiate it into osteoblasts or chondroblasts to form bone or cartilage. Has been started.
[0007]
For example, Patent Document 1 discloses a transplant material in which BMP itself or DNA encoding BMP is carried (applied) on a matrix material (substrate).
[0008]
However, since BMP is a protein, there is a problem that it is easily deactivated, and it has become clear that it is necessary to use a large amount. On the other hand, there is a problem that sufficient bone formation is not efficiently performed even if DNA encoding BMP is used alone. The reason why bone formation is not efficiently performed when DNA encoding BMP is used alone is assumed to be as follows, for example. That is, even when BMP is produced satisfactorily by using DNA encoding BMP and differentiation of undifferentiated mesenchymal cells into osteoblasts or chondroblasts is promoted, these differentiated cells are efficiently used. As a result of not proliferating well, bone formation may not be promoted.
[0009]
[Patent Document 1]
JP-T-2001-505097
[0010]
[Problems to be solved by the invention]
An object of the present invention is to provide an osteogenesis treatment device having excellent osteogenesis ability.
[0011]
[Means for Solving the Problems]
Such an object is achieved by the present inventions (1) to (15) below.
[0012]
(1) An osteogenesis treatment device comprising a nucleic acid comprising a base sequence encoding a bone morphogenetic protein (BMP), an angiogenesis inducing factor, and a biocompatible substrate.
[0013]
ADVANTAGE OF THE INVENTION According to this invention, the bone formation treatment device which has the outstanding bone formation ability can be provided.
[0014]
(2) The angiogenesis inducing factor is at least one of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). Osteogenic treatment device.
[0015]
Since these are excellent in angiogenic ability, the resulting osteogenesis treatment device has particularly high osteogenic ability.
[0016]
(3) The osteogenesis treatment device according to (1) or (2) above, wherein a mixing ratio of the angiogenesis inducing factor and the nucleic acid is 10: 1 to 1: 100 by weight.
[0017]
As a result, new blood vessels are efficiently formed, osteoblasts proliferate efficiently, and bone formation proceeds.
[0018]
(4) The osteogenesis treatment device according to any one of (1) to (3), wherein the bone morphogenic protein is at least one of BMP-2, BMP-4, and BMP-7.
[0019]
BMP-2, BMP-4, and BMP-7 are particularly excellent in the action of inducing differentiation of undifferentiated mesenchymal cells into osteoblasts.
[0020]
(5) The osteogenesis treatment device according to any one of (1) to (4), wherein the nucleic acid includes a base sequence derived from an expression plasmid.
[0021]
Thereby, the expression efficiency of BMP in cells involved in bone formation such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts can be extremely increased.
[0022]
(6) The osteogenesis treatment device according to any one of (1) to (5), wherein the nucleic acid is used in an amount of 1 to 100 μg per mL of the substrate.
Thereby, quicker bone formation can be promoted.
[0023]
(7) The osteogenesis treatment device according to any one of (1) to (6) above, which comprises a vector that holds the nucleic acid.
[0024]
This further improves the efficiency of incorporation of the recombinant plasmid into cells involved in bone formation, and as a result, promotes faster bone formation.
[0025]
(8) The osteogenesis treatment device according to (7), wherein the vector is a non-viral vector.
[0026]
As a result, a relatively large amount of nucleic acid can be easily and reliably supplied to a limited site, and higher patient safety can be ensured.
[0027]
(9) The osteogenesis treatment device according to (8), wherein the non-viral vector is a liposome.
[0028]
Liposomes are composed of components that are close to the components of the cell membrane, so the binding (fusion) to the cell membrane is relatively easy and smooth, such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts of nucleic acids. The efficiency of uptake into cells involved in bone formation can be further improved.
[0029]
(10) The osteogenesis treatment device according to (9), wherein the liposome is a positively charged liposome.
[0030]
The positively charged liposome does not require an operation of encapsulating a nucleic acid therein, which is advantageous for shortening the production time of the osteogenic treatment device.
[0031]
(11) The osteogenesis treatment device according to any one of (7) to (10), wherein a mixing ratio of the vector and the nucleic acid is 1: 1 to 20: 1 by weight.
[0032]
This will increase the efficiency of nucleic acid uptake into cells involved in bone formation, such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts, while preventing increased costs and cytotoxicity. be able to.
[0033]
(12) The osteogenesis treatment device according to any one of (1) to (11), wherein the base is a block body.
[0034]
Accordingly, it is possible to prevent the osteogenesis treatment device from dissipating from the transplant site at an early stage, and it is possible to advance the osteogenesis along the shape of the block body.
[0035]
(13) The osteogenesis treatment device according to any one of (1) to (12), wherein the base is a porous body.
[0036]
As a result, the nucleic acid and angiogenesis-inducing factor can be more easily and reliably supported on the substrate, and cells and blood vessels involved in bone formation such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts are formed. It is easy for cells involved in to enter the substrate, which is advantageous for bone formation.
[0037]
(14) The osteogenesis treatment device according to (13), wherein the porosity of the porous body is 30 to 95%.
[0038]
As a result, while maintaining the mechanical strength of the substrate suitably, cells involved in bone formation such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts, and cells involved in blood vessel formation enter the substrate. Is further facilitated, and the substrate can be used as a more suitable place for bone formation.
[0039]
(15) The osteogenesis treatment device according to any one of (1) to (14), wherein the base is mainly composed of hydroxyapatite or tricalcium phosphate.
[0040]
Since hydroxyapatite and tricalcium phosphate have the same structure as the mineral main component of bone, they have particularly excellent biocompatibility.
[0041]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the osteogenesis treatment device of the present invention will be described in detail.
[0042]
The osteogenesis treatment device of the present invention comprises a nucleic acid containing a base sequence encoding a bone morphogenic protein (BMP), an angiogenesis inducing factor, and a substrate, and transplanted in vivo to perform osteogenesis treatment. Is what you do.
[0043]
As a result of intensive studies in view of the above problems, the present inventor has been involved in cell construction (formation) in order to efficiently proliferate osteoblasts and chondroblasts differentiated from undifferentiated mesenchymal cells. It is thought that it is important to secure a route for supplying various necessary substrates, that is, to form blood vessels for inducing and proliferating osteoblasts and chondroblasts at an early stage around them. The invention has been completed.
[0044]
According to the present invention, the synergistic effect of the combination of a nucleic acid containing a base sequence encoding BMP and an angiogenesis inducer promotes differentiation of undifferentiated mesenchymal cells into osteoblasts and chondroblasts. These differentiated cells can be efficiently proliferated and, as a result, promote bone formation.
[0045]
In addition, an angiogenesis-inducing factor itself directly acts on these differentiated cells (stem cells) and can be expected to proliferate.
[0046]
Here, “bone formation” in the present specification includes both bone formation and cartilage formation, and osteoblasts and chondroblasts (hereinafter referred to as osteoblasts) with respect to undifferentiated mesenchymal cells. ) Refers to bone formation and cartilage formation by inducing differentiation.
[0047]
In addition, “bone formation treatment” means prevention or treatment of a disease requiring formation or compensation of bone tissue or cartilage tissue or improvement of symptoms in the medical field and dental field.
[0048]
In addition, since cDNA is usually used as the base sequence encoding BMP, hereinafter, the base sequence encoding BMP is referred to as “BMP cDNA”.
[0049]
The BMP in the present invention is not particularly limited as long as it has an activity of promoting osteogenesis by inducing differentiation into undifferentiated mesenchymal cells into osteoblasts. For example, BMP-1 , BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-12 (above, homodimers), or heterodimers of these BMPs Or a variant (ie, a protein having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence of a naturally occurring BMP and having the same activity as that of a naturally occurring BMP) Etc. Among these, as BMP, at least one of BMP-2, BMP-4, and BMP-7 is particularly preferable. Since BMP-2, BMP-4, and BMP-7 are particularly excellent in the action of inducing differentiation of undifferentiated mesenchymal cells into osteoblasts, the resulting osteogenesis treatment device has particularly high osteogenic ability. Show.
[0050]
For this reason, the BMP cDNA used in the present invention only needs to include a base sequence capable of producing (expressing) various BMPs as described above. That is, the BMP cDNA is the same as the base sequence encoding a naturally occurring BMP, or one having one or more bases deleted, substituted and / or added in the base sequence encoding a naturally occurring BMP. Can be used. Moreover, you may make it use these things combining 1 type (s) or 2 or more types.
[0051]
Such BMP cDNA can be obtained, for example, according to the methods described in JP-T-2-500141, JP-T-3-503649, JP-T-3-509898 and the like.
[0052]
Further, such a nucleic acid is preferably one containing a nucleotide sequence derived from an expression plasmid, that is, one in which BMP cDNA is incorporated (introduced) into the expression plasmid.
[0053]
Below, what integrated BMP cDNA in the expression plasmid is called "recombinant plasmid", and this recombinant plasmid is demonstrated as a representative of the nucleic acid containing the base sequence which codes BMP cDNA.
[0054]
By using such a recombinant plasmid, undifferentiated mesenchymal cells, inflammatory cells, fibroblasts, and the like that have incorporated the plasmids (hereinafter collectively referred to as “cells involved in bone formation”). The expression efficiency of BMP can be made extremely high.
[0055]
The expression plasmid can be selected from those widely used in the field of genetic engineering technology. For example, one or more of pCAH, pSC101, pBR322, pUC18 and the like can be used in combination.
[0056]
In addition, a base sequence (DNA fragment) that appropriately controls the expression of BMP can be appropriately introduced into this recombinant plasmid.
[0057]
As a method for incorporating various base sequences including BMP cDNA into the expression plasmid, a known method can be used.
[0058]
Here, FIG. 1 shows an example of a recombinant plasmid (chimeric DNA).
The recombinant plasmid shown in FIG. 1 is obtained by introducing BMP-2 cDNA into the expression plasmid pCAH.
[0059]
This recombinant plasmid contains a DNA fragment that is resistant to Amp (ampicillin), a DNA fragment containing an enhancer promoter derived from cytomegalovirus (CMV), and a region derived from SV40 in the downstream region of BMP-2 cDNA. And a DNA fragment containing a transcription termination signal.
[0060]
The amount of the recombinant plasmid (nucleic acid) to be used is not particularly limited, but is preferably about 1 to 100 μg, more preferably about 10 to 70 μg per 1 mL of the substrate volume described later. If too little recombinant plasmid is used, rapid bone formation may not be promoted. On the other hand, even if the amount of the recombinant plasmid used exceeds the upper limit, no further increase in the effect can be expected.
[0061]
The angiogenesis-inducing factor in the present invention is not particularly limited as long as it can promote angiogenesis. For example, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (bFGF) Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), Granulocyte Macrophage Colon-Stimulating Factor (GFS) : G-CSF), macrophage colony stimulating factor (Ma crop-Colony Stimulating Factor (M-CSF), Stem Cell Factor (SCF), Angiopoietin-1, Angiopoietin-2, liponuclease-like protein, nicotinamide proprotein E (Prostaglandin E ₁ , Prostaglandin E ₂ , Prostaglandin E ₃ ), Proline derivatives, cyclic AMP derivatives such as dibutyl cyclic AMP (dBcAMP), and the like, and one or more of these can be used in combination. Among these, the angiogenesis inducing factor is particularly preferably at least one of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). . Since these are excellent in angiogenic ability, the resulting osteogenesis treatment device has particularly high osteogenic ability.
[0062]
The amount of angiogenesis-inducing factor used is appropriately set depending on the type and the like, and is not particularly limited. It is preferable that it is about 1: 1 to 1: 100. If the amount of angiogenesis-inducing factor used is too small, depending on the type of angiogenesis-inducing factor, new blood vessels may not be formed efficiently, and osteoblasts may not be able to proliferate sufficiently. On the other hand, even if the amount of angiogenesis-inducing factor used exceeds the upper limit, no further increase in effect can be expected.
[0063]
In addition, the osteogenesis treatment device of the present invention has a biocompatible substrate. This substrate serves as a field for bone formation by osteoblasts differentiated from undifferentiated mesenchymal cells.
[0064]
The form of the substrate is preferably a block body (lump). The block body (for example, a sintered body) has shape stability, and when an osteogenesis treatment device is implanted in a living body, the osteogenesis treatment device can be prevented from dissipating from the implantation site at an early stage. In addition, since bone formation can proceed along the shape of the block body, it is particularly effective when the transplant site is a relatively large bone defect or the like.
[0065]
The form of the substrate may be appropriately selected according to the application site (transplantation site) of the osteogenesis treatment device. For example, it may be in the form of powder, granules, pellets (small lumps), etc. Good. In the case of using the substrate in such a form, a composition mixed with a recombinant plasmid (nucleic acid) and an angiogenesis inducing factor can be used as an osteogenesis treatment device. It can be used by filling (packing).
[0066]
The substrate is preferably porous (porous body). By using a porous material as a substrate, the recombinant plasmid (nucleic acid) and angiogenesis-inducing factor can be more easily and reliably carried on the substrate, and cells involved in bone formation or cells involved in blood vessel formation (For example, vascular endothelial cells and the like) easily enter the substrate, which is advantageous for bone formation.
[0067]
In this case, the porosity is not particularly limited, but is preferably about 30 to 95%, more preferably about 55 to 90%. By setting the porosity within the above range, it is possible to further facilitate the entry of cells involved in bone formation and cells involved in blood vessel formation into the substrate while maintaining the mechanical strength of the substrate suitably. It can be a suitable place for bone formation.
[0068]
Further, the constituent material of the substrate is not particularly limited as long as it has biocompatibility. For example, hydroxyapatite, fluorapatite, carbonate apatite, dicalcium phosphate, tricalcium phosphate, tetracalcium phosphate , Calcium phosphate compounds such as octacalcium phosphate, ceramic materials such as alumina, titania, zirconia, yttria, various metal materials such as titanium or titanium alloys, stainless steel, Co-Cr alloys, Ni-Ti alloys, etc. 1 type or 2 types or more of these can be used in combination.
[0069]
Among these, as a constituent material of the substrate, a ceramic material (so-called bioceramics) such as a calcium phosphate compound, alumina, zirconia is preferable, and a material mainly composed of hydroxyapatite or tricalcium phosphate is particularly preferable.
[0070]
Since hydroxyapatite and tricalcium phosphate have the same structure as the mineral main component of bone, they have particularly excellent biocompatibility. Moreover, since it has both positive and negative charges, especially when using liposome as a vector (this point will be described in detail later), this liposome can be stably supported on a substrate for a long time. . As a result, the recombinant plasmid (nucleic acid) adsorbed or encapsulated in the liposome is also stably retained on the substrate for a long time, contributing to more rapid bone formation. Moreover, since it has high affinity with osteoblasts, it is preferable for maintaining new bone.
[0071]
Such a substrate can be produced (manufactured) by various methods. The case where a porous block body made of a ceramic material is produced as an example will be described as an example. Such a porous block body is made of, for example, a slurry containing a ceramic material powder and transplanted into a bone defect or the like. It can be produced by obtaining a molded body formed into a shape corresponding to a part by, for example, compression molding or the like, and sintering (firing) the molded body.
[0072]
The osteogenesis treatment device as described above can be produced (manufactured) by bringing a recombinant plasmid (nucleic acid) and an angiogenesis inducing factor into contact with a substrate. Specifically, the osteogenesis treatment device is, for example, a liquid (solution or suspension) containing either one of a recombinant plasmid and an angiogenesis inducer, or both a recombinant plasmid and an angiogenesis inducer. It can be easily produced by supplying a liquid containing a liquid to the substrate or immersing the substrate in these liquids.
[0073]
When using a powder, granule, pellet or the like as the substrate, for example, the osteogenesis treatment device can be obtained by molding a kneaded material obtained by kneading the substrate, the binder and the liquid as described above. It can also be produced.
[0074]
When such an osteogenesis treatment device is embedded (applied) at a transplant site such as a bone defect, for example, cells involved in osteogenesis in the vicinity of the osteogenesis treatment device are incorporated into the recombinant plasmid ( Nucleic acid). In these cells, BMP is sequentially produced using the recombinant plasmid as a template, and this BMP induces differentiation of undifferentiated mesenchymal cells into osteoblasts. At this time, new blood vessels are actively formed inside the substrate (that is, around the osteoblasts) by the action of the angiogenesis-inducing factor, and various kinds of osteoblasts necessary for the growth of osteoblasts via these blood vessels are formed. Substrate is supplied. Thereby, osteoblasts proliferate efficiently and as a result, bone formation proceeds.
[0075]
Furthermore, such osteogenic treatment device preferably includes a vector. This vector has a function of retaining a recombinant plasmid (nucleic acid) and promoting uptake of the recombinant plasmid into cells involved in bone formation. By using the vector, the efficiency of incorporation of the recombinant plasmid into cells involved in bone formation is further improved, and as a result, more rapid bone formation is promoted.
[0076]
As the vector, any of vectors derived from viruses such as non-virus-derived vectors (that is, non-virus-derived vectors), adenovirus vectors, and retrovirus vectors may be used, but non-virus-derived vectors are preferably used. . By using a non-viral vector, a relatively large amount of recombinant plasmid can be supplied easily and reliably to a limited site, and since it does not cause infection, higher patient safety is ensured. There is also an advantage of being able to. Furthermore, the method using a non-viral vector is an ex vivo method using a viral vector or cell, such as an operation for introducing a nucleic acid into a viral vector or cell, an operation for propagating a viral vector or cell into which a nucleic acid has been introduced, etc. Although it is necessary, since these operations are not required, it is excellent in that time and labor can be reduced.
[0077]
Various vectors can be used as the non-viral vector, but it is preferable to use a liposome (lipid membrane). Since the liposome is composed of components close to the constituent components of the cell membrane, the binding (fusion) to the cell membrane is relatively easy and smooth. For this reason, it is possible to further improve the efficiency of uptake of the recombinant plasmid into cells involved in bone formation.
[0078]
As the liposome, for example, a positively charged liposome that adsorbs the recombinant plasmid on the surface, a negatively charged liposome that encloses the recombinant plasmid inside, and the like can be used. These liposomes can be used alone or in combination.
[0079]
The positively charged liposome is mainly composed of a polycationic lipid such as DOSPA (2,3-dioleoyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanamine trifluorate). As the positively charged liposome, for example, a commercially available product such as “SuperFect” manufactured by QIAGEN can be used.
[0080]
On the other hand, negatively charged liposomes are phospholipids such as 3-sn-phosphatidylcholine, 3-sn-phosphatidylserine, 3-sn-phosphatidylethanolamine, 3-sn-phosphatidylethanolamine, or derivatives thereof. Is the main thing.
[0081]
Moreover, you may make it add the additive which stabilizes lipid membranes, such as cholesterol, for example to these liposomes.
[0082]
In addition, as the liposome, it is particularly preferable to use a positively charged liposome. The positively charged liposome does not require an operation for encapsulating the recombinant plasmid therein, and thus is advantageous in shortening the production time of the osteogenic treatment device.
[0083]
The amount of the vector to be used is appropriately set depending on the type and the like, and is not particularly limited. However, the mixing ratio of the vector and the recombinant plasmid (nucleic acid) is preferably about 1: 1 to 20: 1 by weight, More preferably, it is about 2: 1 to 10: 1. If the amount of the vector used is too small, the efficiency of incorporation of the recombinant plasmid into the cells involved in bone formation may not be sufficiently increased depending on the type of vector. On the other hand, even if the amount of the vector to be used is increased beyond the upper limit, not only an increase in the effect cannot be expected, but also cytotoxicity may occur. Moreover, it causes an increase in cost, which is not preferable.
[0084]
The preferred embodiment of the osteogenesis treatment device of the present invention has been described above, but the present invention is not limited to this.
[0085]
In the above embodiment, a recombinant plasmid in which BMP cDNA is incorporated into an expression plasmid as a nucleic acid containing a base sequence encoding bone morphogenic protein (BMP) has been described as a representative. However, the base sequence encoding BMP in the present invention is The nucleic acid to be contained may be, for example, BMP cDNA (not incorporated into an expression plasmid), BMP mRNA, or those obtained by adding an arbitrary base thereto.
[0086]
【Example】
Next, specific examples of the present invention will be described.
[0087]
(Example)
1. Preparation of recombinant plasmid
By a known method, human BMP-2 cDNA (base sequence encoding human BMP-2) and a desired base sequence were incorporated into an expression plasmid to obtain a recombinant plasmid as shown in FIG.
[0088]
And this recombinant plasmid was propagated as follows.
First, at room temperature, the recombinant plasmid was added to 200 μL of a suspension of DH5α (Competent Bacteria).
[0089]
Next, this mixed solution was added to the LB agar medium and cultured at 37 ° C. for 12 hours. Next, after this culture was completed, a relatively large colony was selected from the colonies grown on the LB agar medium, and this was transplanted to an LB agar medium containing Amp (ampicillin) and further cultured at 37 ° C. for 12 hours. .
[0090]
Thereafter, the cell membrane of DH5α grown on the LB agar medium containing Amp was broken, and the recombinant plasmid was purified and separated from the solution.
[0091]
2. Production of hydroxyapatite porous sintered body (substrate)
Hydroxyapatite was synthesized by a known wet synthesis method to obtain a hydroxyapatite slurry.
[0092]
This hydroxyapatite slurry was spray-dried to obtain a powder having an average particle size of about 15 μm. Thereafter, the powder was calcined at 700 ° C. for 2 hours, and then pulverized to an average particle size of about 12 μm using a general-purpose pulverizer. In order to foam the mixed liquid containing the pulverized hydroxyapatite powder and the water-soluble polymer, the mixture was stirred to obtain a paste. The hydroxyapatite powder and the water-soluble polymer were mixed at a weight ratio of 5: 6.
[0093]
This paste-like kneaded product was put in a mold and dried at 80 ° C. to gel the water-soluble polymer, thereby producing a molded product. The molded body was processed into a disk shape having a diameter of 10 mm and a thickness of 3 mm (volume: about 0.24 mL) using a processing machine such as a general-purpose lathe.
[0094]
This disk-shaped molded body was fired in the atmosphere at 1200 ° C. for 2 hours to obtain a hydroxyapatite porous sintered body.
[0095]
The hydroxyapatite porous sintered body had a porosity of 70%. This measurement was performed by the Archimedes method.
[0096]
3. Fabrication of osteogenic treatment device
Phosphate buffer containing recombinant plasmid, phosphate buffer containing basic fibroblast growth factor (bFGF), an angiogenesis inducing factor, and positively charged liposome (QIAGEN, “SuperFect”, vector) )), And the hydroxyapatite porous sintered body is prepared so that the recombinant plasmid is 10 μg, the basic fibroblast growth factor (bFGF) is 1 μg, and the positively charged liposome is 40 μg. Impregnated.
Thereby, the osteogenesis treatment device was obtained.
[0097]
(Comparative Example 1)
An osteogenesis treatment device was produced in the same manner as in the above example except that the basic fibroblast growth factor was not used.
[0098]
(Comparative Example 2)
An osteogenesis treatment device was produced in the same manner as in the above example except that the recombinant plasmid and the positively charged liposome were not used.
[0099]
(Comparative Example 3)
An osteogenesis treatment device was prepared in the same manner as in the above example, except that the recombinant plasmid and basic fibroblast growth factor were not used.
[0100]
<Evaluation>
1. Evaluation experiment
72 rabbits (average weight 3.0 kg) were prepared. The following operations were performed on each rabbit.
[0101]
First, the rabbit was anesthetized by intravenous administration of 25 mg / kg pentobarbital sodium (Abbott Laboratories, “Nembutal”).
[0102]
Next, an incision was made in the scalp of a rabbit, and the skin was raised as a flap having a width of 2.5 cm and a length of 3.0 cm with the tail side as a stem.
[0103]
Next, an incision of 2 to 3 mm was made in the exposed periosteum, and a periosteum exfoliant was applied to this part, and a part having a diameter of about 3 mm was exfoliated to expose the skull.
[0104]
Next, the midline of the exposed skull was opened using a skull burr, and the skull was removed to just above it so that the dura mater was preserved. In addition, the thickness of the skull was about 3 mm, and the diameter of the craniotomy part was about 1.2 cm.
[0105]
Next, the rabbits that had undergone craniotomy were divided into 4 groups of 18 each, and each of the rabbits in the first group had the osteogenic treatment device of the example, and each rabbit in the second group had The osteogenic treatment device of Comparative Example 1 was compared with each of the rabbits in the third group, and the osteogenic treatment device of Comparative Example 2 was compared with each of the rabbits in Comparative Group 2, respectively. After implantation of the osteogenic treatment device of Example 3, the flap was returned to its original position and sutured.
[0106]
Then, each rabbit that had undergone surgery was raised in an individual cage.
[0107]
2. Evaluation results
At 3, 6, and 9 weeks after the operation, 6 rabbits in each group were sacrificed by overdosing the same anesthetic as described above.
[0108]
Thereafter, the skull was excised as a lump together with the skin directly above, and the collected tissue was immediately immersed in 10% neutral buffered formalin solution and fixed, and then embedded in a polyester resin. Next, the tissue embedded in the polyester resin was thinly cut and polished so as to have a thickness of 50 μm, and then subjected to colle-HE staining. Thereby, a tissue specimen was obtained.
[0109]
About each obtained tissue specimen, the bone formation rate was measured as follows, respectively. That is, each tissue specimen was photographed with a stereomicroscope system SZX-12 (Olympus) with a digital camera (DP-12). Next, using photoshop_ver4.0 (manufactured by Adobe), a new bone portion is extracted from the photographed image data by digital processing, and further extracted by an image analysis method using a SCION image (manufactured by Scion). The bone formation rate was calculated by measuring the area of the newly formed bone part.
[0110]
The bone formation rate was measured in a range of 5 mm × 3 mm thickness of the hydroxyapatite porous sintered body from the end in the surface direction (direction perpendicular to the thickness direction) of the hydroxyapatite porous sintered body. .
The results are shown in Table 1.
[0111]
[Table 1]

[0112]
As shown in Table 1, the bone formation rate of the osteogenesis treatment device of the example (bone formation treatment device of the present invention) is compared with the bone formation rate of the osteogenesis treatment device of Comparative Examples 1 to 3, Some were comparable or lower after 3 weeks and 6 weeks, but after 9 weeks it was significantly higher than either.
[0113]
As described above, it has been clarified that an osteogenesis treatment device having extremely excellent osteogenic ability can be obtained by using the recombinant plasmid and basic fibroblast growth factor (bFGF) in combination.
[0114]
Further, as a substrate, instead of the hydroxyapatite porous sintered body, a bone formation treatment device was produced using a tricalcium phosphate porous sintered body, and the same evaluation experiment as described above was conducted. Similar evaluation results were obtained.
[0115]
Further, as a base sequence encoding BMP, instead of a base sequence encoding human BMP-2, human BMP-1, human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP- 6, a base sequence encoding human BMP-7, human BMP-8, human BMP-9 or human BMP-12, or any combination thereof, to produce an osteogenesis treatment device, and the same evaluation experiment as described above As a result of the evaluation, almost the same evaluation results as in the above example were obtained.
[0116]
In addition, instead of basic fibroblast growth factor (bFGF) as an angiogenesis inducing factor, vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF), or any combination thereof can be used as an osteogenesis treatment device. As a result of manufacturing and performing an evaluation experiment similar to the above, an evaluation result almost the same as that of the above example was obtained.
[0117]
【The invention's effect】
As described above, according to the present invention, extremely rapid bone formation is possible, and it is possible to contribute to early bone formation treatment.
[0118]
In particular, in the present invention, by using an angiogenesis-inducing factor in combination, new blood vessels are actively formed around osteoblasts, and osteoblasts proliferate efficiently, resulting in rapid bone formation. .
[0119]
For this reason, it is not necessary to perform free bone grafting in various bone formation treatments, and a bone collecting part is not necessary, so that safer, more reliable, and rational surgery is possible.
[0120]
Because of this, it is possible to shorten the operation time and hospitalization period, and it is possible to reduce the total medical cost, improve the treatment quality, and improve the patient's QOL.
[0121]
In addition, the combined use of a nucleic acid-retaining vector improves the efficiency of nucleic acid uptake into cells involved in bone formation such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts. More rapid bone formation is achieved, and the effect is further improved.
[0122]
In addition, by using a block body as a base, bone formation proceeds well along the shape of the base, which is effective when the transplant site is a relatively large bone defect or the like.
[0123]
In addition, by using a porous material specialized for gene therapy as a substrate, it is possible to more easily and reliably carry the nucleic acid and angiogenesis-inducing factor on the substrate, as well as cells involved in various bone formations and Cells that are involved in angiogenesis can easily enter the substrate, which is advantageous for bone formation.
[0124]
In addition, the osteogenesis treatment device of the present invention is easy to store, handle, process during surgery, and the like.
[Brief description of the drawings]
FIG. 1 is a genetic map showing an example of a recombinant plasmid.

Claims (6)

Biocompatible substrate mainly composed of hydroxyapatite or tricalcium phosphate having both positive and negative charges, a nucleic acid containing a base sequence encoding bone morphogenic protein (BMP) and a base sequence derived from an expression plasmid, and angiogenesis induction An agent and at least one of positively charged liposomes and negatively charged liposomes,
The liposome is supported on the substrate having both positive and negative charges, and the liposome holds the nucleic acid,
The nucleic acid is 1 to 100 μg per mL of the substrate, the angiogenesis factor and the nucleic acid are in a weight ratio of 10: 1 to 1: 100, and the liposome and the nucleic acid are in a weight ratio of 1: 1 to 1. An osteogenesis treatment device characterized by being formulated at 20: 1. The bone formation treatment according to claim 1, wherein the angiogenesis inducing factor is at least one of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). device. The osteogenesis treatment device according to claim 1 or 2, wherein the bone morphogenic protein is at least one of BMP-2, BMP-4, and BMP-7. The osteogenesis treatment device according to claim 1, wherein the base body is a block body. The osteogenesis treatment device according to any one of claims 1 to 4, wherein the substrate is a porous body. The osteogenesis treatment device according to claim 5, wherein the porosity of the porous body is 30 to 95%.

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