JP2004123610A - Osteogenic treatment device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an osteogenic treatment device having excellent osteogenesis ability. <P>SOLUTION: This osteogenic treatment device comprises a nucleic acid containing a base sequence encoding a bone morphogenetic protein (BMP), a vector and a substrate body and is transplanted in a living body to carry out osteogenic treatment. The bone morphogenetic protein is preferably BMP-2 and the nucleic acid preferably contains an expression plasmid derived base sequence. The vector is preferably a positively charged liposome being a vector derived from a nonvirus. The form of its substrate body is preferably a block body, especially a porous body having 30-95% porosity. The constituent material of the substrate body is a material consisting essentially of hydroxyapatite or tricalcium phosphate. <P>COPYRIGHT: (C)2004,JPO

Description

【０１００】
表１に示すように、実施例の骨形成治療デバイスは、極めて骨形成能に優れるものであった。
これに対し、比較例の骨形成治療デバイスは、骨形成能に劣るものであった。
【０１０１】
このように、ハイドロキシアパタイト多孔質焼結体と、組換えプラスミドと、正荷電リポソーム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。
【０１０２】
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。
【０１０３】
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、前記実施例とほぼ同様の評価結果が得られた。
【０１０４】
【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。
【０１０５】
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。
【０１０６】
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。
【０１０７】
特に、本発明では、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。
【０１０８】
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。
【０１０９】
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、核酸およびベクターを、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が基体内に侵入し易くなり、骨形成にとって有利である。
【０１１０】
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 OF THE INVENTION
The present invention relates to an osteogenic treatment device.
[0002]
[Prior art]
In the medical field, research on so-called artificial bones, artificial skins, and artificial organs has attracted attention, and new challenges have been made from various viewpoints even in clinical settings. Also show a great extension.
[0003]
In particular, with regard to allogeneic transplantation, there is a problem that the state in which the demand greatly exceeds the supply chronically persists, and there is a problem such as unknown infectious disease.
[0004]
In order to avoid these problems, development of artificially produced artificial tissues and artificial organs is expected as an alternative to such transplantation of allogeneic tissues. In this regard, the same applies to bone grafts.
[0005]
Many researchers have elucidated the process of bone formation and repair, and in addition to discovering, identifying, and isolating osteoinductive factors, which are key to their control, it has become possible to generate osteoinductive factors by genetic engineering techniques. Knowledge of its mechanism of action has also been accumulated.
[0006]
As this osteoinductive factor, bone morphogenic protein (BMP) belonging to the super family of transforming growth factor b (TGFb) has attracted many researchers' attention. This BMP is an active protein that acts on undifferentiated mesenchymal cells in subcutaneous tissue or muscle tissue, differentiates it into osteoblasts and chondroblasts, and forms bone or cartilage. Has been started.
[0007]
For example, Patent Document 1 discloses an implant material in which BMP itself or DNA encoding BMP is carried (applied) on a matrix material (carrier).
[0008]
However, since BMP is a protein, it is easily inactivated, and even when DNA encoding BMP is used alone, the uptake into the undifferentiated mesenchymal cells is extremely low, and sufficient bone formation is not efficiently performed. There is a problem that is not done.
[0009]
[Patent Document 1]
JP 2001-505097 A
[0010]
[Problems to be solved by the invention]
An object of the present invention is to provide a bone formation treatment device having excellent bone formation ability.
[0011]
[Means for Solving the Problems]
Such an object is achieved by the following (1) to (12) of the present invention.
[0012]
(1) a nucleic acid comprising a base sequence encoding a bone morphogenetic protein (BMP);
A vector holding the nucleic acid,
An osteogenic treatment device comprising: a biocompatible substrate.
[0013]
According to the present invention, it is possible to provide an osteogenic treatment device having excellent osteogenic ability.
[0014]
(2) The bone formation treatment device according to (1), wherein the bone morphogenetic protein is BMP-2.
[0015]
BMP-2 is particularly excellent in inducing the differentiation of undifferentiated mesenchymal cells into osteoblasts.
[0016]
(3) The device for treating osteogenesis according to the above (1) or (2), wherein the nucleic acid contains a base sequence derived from an expression plasmid.
[0017]
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.
[0018]
(4) The bone formation treatment device according to any one of (1) to (3), wherein the nucleic acid is used in an amount of 1 to 100 μg per 1 mL of the volume of the substrate.
Thereby, more rapid bone formation can be promoted.
[0019]
(5) The osteogenesis treatment device according to any one of (1) to (4), wherein the vector is a non-virus-derived vector.
[0020]
As a result, a relatively large amount of nucleic acid can be easily and reliably supplied to the localized site, and higher safety of the patient can be ensured.
[0021]
(6) The osteogenic treatment device according to (5), wherein the non-virus-derived vector is a liposome.
[0022]
Since liposomes are composed of components close to the components of cell membranes, they can be relatively easily and smoothly bonded (fused) to cell membranes, and can be used for undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts. The efficiency of uptake into cells involved in such bone formation can be further improved.
[0023]
(7) The osteogenic treatment device according to (6), wherein the liposome is a positively charged liposome.
[0024]
Positively charged liposomes do not require an operation of encapsulating nucleic acids therein, and are therefore advantageous in shortening the time required for producing an osteogenic treatment device.
[0025]
(8) The osteogenesis treatment device according to any one of (1) to (7), wherein a mixing ratio of the vector and the nucleic acid is 1: 1 to 20: 1 by weight.
[0026]
In this way, the efficiency of incorporation of nucleic acids into cells involved in bone formation, such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts, is sufficiently increased, while preventing cost increase and occurrence of cytotoxicity. be able to.
[0027]
(9) The bone formation treatment device according to any one of (1) to (8), wherein the base is a block.
[0028]
Thereby, it is possible to prevent the osteogenesis treatment device from escaping from the implantation site at an early stage, and to allow the osteogenesis to progress along the shape of the block body.
[0029]
(10) The bone formation treatment device according to any one of (1) to (9), wherein the base is a porous body.
[0030]
As a result, the nucleic acid and the vector can be more easily and reliably carried on the substrate, and cells involved in bone formation such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts enter the substrate. It is easier for bone formation.
[0031]
(11) The bone formation treatment device according to (10), wherein the porosity of the porous body is 30 to 95%.
[0032]
This makes it easier to infiltrate cells involved in bone formation, such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts, into the substrate while suitably maintaining the mechanical strength of the substrate. A more suitable site for bone formation can be obtained.
[0033]
(12) The bone formation treatment device according to any one of (1) to (11), wherein the substrate is mainly composed of hydroxyapatite or tricalcium phosphate.
[0034]
Hydroxyapatite and tricalcium phosphate have particularly excellent biocompatibility because they have the same structure as the inorganic main component of bone.
[0035]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the osteogenic treatment device of the present invention will be described in detail.
[0036]
The osteogenesis treatment device of the present invention includes a nucleic acid containing a base sequence encoding a bone morphogenetic protein (BMP), a vector, and a substrate, and is used for transplantation into a living body to perform osteogenesis treatment. is there.
[0037]
Here, the term “osteogenesis” in the present specification includes both osteogenesis and chondrogenesis, and osteoblasts and chondroblasts (hereinafter, osteoblasts are representative of undifferentiated mesenchymal cells). ) Refers to osteogenesis and cartilage formation by inducing differentiation.
[0038]
The term “bone formation treatment” refers to preventing or treating a disease that requires formation or replacement of bone or cartilage tissue or improving symptoms in the medical and dental fields.
[0039]
In addition, since a cDNA is usually used as a base sequence encoding BMP, the base sequence encoding BMP is hereinafter referred to as “BMP cDNA”.
[0040]
The BMP in the present invention is not particularly limited as long as it has an activity to promote bone formation by inducing differentiation of undifferentiated mesenchymal cells into osteoblasts, and is not particularly limited. , BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-12 (or more, homodimers), or heterodimers of these BMPs Or a variant (ie, a protein having an amino acid sequence in which one or more amino acids have been deleted, substituted and / or added in the amino acid sequence of a naturally-occurring BMP, and having the same activity as a naturally-occurring BMP) And the like. Among them, BMP-2 is particularly preferable as BMP. Since BMP-2 is particularly excellent in inducing the differentiation of undifferentiated mesenchymal cells into osteoblasts, the resulting osteogenic treatment device exhibits particularly high osteogenic ability.
[0041]
For this reason, the BMP cDNA used in the present invention may be any as long as it contains a base sequence capable of producing (expressing) various BMPs as described above. That is, the BMP cDNA is the same as the nucleotide sequence encoding a naturally-occurring BMP, or one in which one or more bases have been deleted, substituted and / or added in the nucleotide sequence encoding a naturally-occurring BMP. I just need. These may be used alone or in combination of two or more.
[0042]
Such a BMP cDNA can be obtained, for example, according to the method described in JP-A-2-500241, JP-A-3-503649, JP-A-3-505098, and the like.
[0043]
Further, such a nucleic acid preferably contains a base sequence derived from an expression plasmid, that is, a nucleic acid in which BMP cDNA has been incorporated (introduced) into the expression plasmid.
[0044]
Hereinafter, the one in which BMP cDNA has been incorporated into an expression plasmid is referred to as a “recombinant plasmid”, and this recombinant plasmid will be described as a representative nucleic acid containing a base sequence encoding BMP cDNA.
[0045]
By using such a recombinant plasmid, undifferentiated mesenchymal cells, inflammatory cells, fibroblasts, and the like incorporating the recombinant plasmid (hereinafter, these are collectively referred to as "cells involved in bone formation"). BMP expression efficiency can be extremely high.
[0046]
The expression plasmid can be selected from those widely used in the field of genetic engineering technology. For example, one or a combination of two or more of pCAH, pSC101, pBR322, pUC18 and the like can be used.
[0047]
In addition, a base sequence (DNA fragment) that appropriately controls BMP expression can be appropriately introduced into this recombinant plasmid.
[0048]
Known methods can be used for incorporating various base sequences including BMP cDNA into the expression plasmid.
[0049]
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 pCAH which is an expression plasmid.
[0050]
This recombinant plasmid contains a DNA fragment showing resistance to Amp (ampicillin), a DNA fragment containing an enhancer promoter derived from cytomegalovirus (CMV), and an SV40-derived DNA downstream of BMP-2 cDNA. And a DNA fragment containing a transcription termination signal.
[0051]
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 volume of the substrate described below. If the amount of the recombinant plasmid used is too small, 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.
[0052]
The 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.
[0053]
As the vector, any of non-virus-derived vectors (that is, non-virus-derived vectors), adenovirus vectors, and virus-derived vectors such as retrovirus vectors may be used, but non-virus-derived vectors are preferably used. . By using a non-viral-derived vector, a relatively large amount of a recombinant plasmid can be easily and reliably supplied to a localized site, and higher safety of a patient is ensured because no infection occurs. There is also the advantage that it can be done. Furthermore, a method using a non-virus-derived vector, such as an ex vivo method using a virus vector or a cell, involves an operation of introducing a nucleic acid into a virus vector or a cell, and an operation of growing a virus vector or a cell into which the nucleic acid has been introduced. In contrast to the necessity, these operations are not required, which is excellent in that time and labor can be reduced.
[0054]
Various vectors can be used as the non-virus-derived vector, but liposomes (lipid membranes) are preferably used. Since liposomes are composed of components close to those of the cell membrane, binding (fusion) to the cell membrane is relatively easy and smooth. For this reason, the efficiency of incorporation of the recombinant plasmid into cells involved in bone formation can be further improved.
[0055]
As the liposome, for example, a positively charged liposome having a surface on which the recombinant plasmid is adsorbed, a negatively charged liposome having a structure in which the recombinant plasmid is encapsulated therein, and the like can be used. These liposomes can be used alone or in combination.
[0056]
Positively charged liposomes mainly contain, for example, polycationic lipids such as DOSPA (2,3-dioleoxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate). As the positively charged liposome, for example, a commercially available product such as “SuperFect” manufactured by QIAGEN can be used.
[0057]
On the other hand, negatively charged liposomes are, for example, phospholipids such as 3-sn-phosphatidylcholine, 3-sn-phosphatidylserine, 3-sn-phosphatidylethanolamine, 3-sn-phosphatidylethanolamine, or derivatives thereof. Mainly.
[0058]
In addition, an additive for stabilizing a lipid membrane such as cholesterol may be added to these liposomes.
[0059]
In addition, it is particularly preferable to use a positively charged liposome as the liposome. Positively charged liposomes do not require an operation of enclosing the recombinant plasmid therein, and thus are advantageous in shortening the time required for producing an osteogenic treatment device.
[0060]
The amount of the vector used is not particularly limited, but the mixing ratio of the vector and the recombinant plasmid (nucleic acid) is preferably about 1: 1 to 20: 1 by weight, and is preferably about 2: 1 to 10: 1. Is more preferable. If the amount of the vector used is too small, the efficiency of incorporation of the recombinant plasmid into cells involved in bone formation may not be sufficiently increased depending on the type of the vector and the like. On the other hand, if the amount of the vector to be used is increased beyond the upper limit, not only the effect cannot be further increased but also cytotoxicity may occur. In addition, the cost is undesirably increased.
[0061]
Further, the osteogenic 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.
[0062]
The form of the substrate is preferably a block (lump). The block body (e.g., a sintered body) has shape stability, and can prevent the osteogenic treatment device from escaping from the implantation site early when the osteogenic treatment device is implanted in a living body. In addition, bone formation can proceed along the shape of the block body, which is particularly effective when the transplantation site is a relatively large bone defect or the like.
[0063]
The form of the base may be appropriately selected depending on the application site (implantation site) of the osteogenesis treatment device, and may be, for example, a powder, a granule, a pellet (small block), or the like. Good. When a substrate having such a form is used, a composition mixed with a recombinant plasmid (nucleic acid) and a vector can be used as an osteogenesis treatment device, and the osteogenesis treatment device is filled in a bone defect. (Stuffing).
[0064]
The substrate is preferably a porous one (porous body). By using a porous body as a substrate, a recombinant plasmid (nucleic acid) and a vector can be more easily and reliably carried on the substrate, and cells involved in bone formation can easily enter the substrate, and It is advantageous for formation.
[0065]
In this case, the porosity is not particularly limited, but is preferably about 30 to 95%, and more preferably about 55 to 90%. When the porosity is in the above range, the cells involved in bone formation can be more easily penetrated into the substrate while suitably maintaining the mechanical strength of the substrate. can do.
[0066]
The constituent material of the substrate is not particularly limited as long as it has biocompatibility, and examples thereof include, for example, hydroxyapatite, fluorapatite, carbonate apatite, dicalcium phosphate, tricalcium phosphate, and tetracalcium phosphate. Calcium phosphate compounds such as octacalcium phosphate, ceramic materials such as alumina, titania, zirconia, and yttria, various metal materials such as titanium or titanium alloy, stainless steel, Co-Cr alloy, and Ni-Ti alloy. And one or more of these can be used in combination.
[0067]
Among these, as a constituent material of the base, a ceramic material (so-called bioceramic) such as a calcium phosphate compound, alumina, or zirconia is preferable, and particularly, a material mainly containing hydroxyapatite or tricalcium phosphate is preferable.
[0068]
Hydroxyapatite and tricalcium phosphate have particularly excellent biocompatibility because they have the same structure as the inorganic main component of bone. In addition, since the liposome has both positive and negative charges, particularly when a liposome is used as a vector, the liposome can be stably supported on the substrate for a long time. As a result, the recombinant plasmid (nucleic acid) adsorbed or encapsulated in the liposome is stably retained on the substrate for a long time, and contributes to more rapid bone formation. In addition, since it has a high affinity for osteoblasts, it is preferable for maintaining new bone.
[0069]
Such a substrate can be produced (manufactured) by various methods. As an example, a case in which a porous block made of a ceramic material is produced as a base will be described. For example, such a porous block can be used to transfer a slurry containing a powder of a ceramic material to a bone defect or the like. It can be manufactured by obtaining a molded body formed by, for example, compression molding or the like into a shape corresponding to the site, and sintering (firing) the molded body.
[0070]
The bone formation treatment device as described above can be prepared (manufactured) by bringing a recombinant plasmid (nucleic acid) and a vector into contact with a substrate. Specifically, for example, the osteogenesis treatment device comprises, as a substrate, a liquid (solution or suspension) containing either the recombinant plasmid and the vector, or a liquid containing both the recombinant plasmid and the vector. It can be easily prepared by supplying or immersing the substrate in these liquids.
[0071]
In the case where a powder, granule, pellet, or the like is used as the substrate, for example, a bone-forming device is prepared by molding a kneaded product obtained by kneading a substrate, a binder, and a liquid as described above. You can also.
[0072]
When such an osteogenesis treatment device is implanted (applied) at a transplant site such as a bone defect, cells involved in osteogenesis existing near the osteogenesis treatment device will have a recombinant plasmid ( Nucleic acid). In these cells, BMP is produced sequentially using the recombinant plasmid as a template, and the BMP induces the differentiation of undifferentiated mesenchymal cells into osteoblasts, and as a result, bone formation proceeds.
[0073]
In particular, in the present invention, by using a vector in combination, the efficiency of incorporation of the recombinant plasmid (nucleic acid) into cells involved in bone formation is improved, and as a result, bone formation is performed more rapidly.
[0074]
The preferred embodiment of the osteogenic treatment device of the present invention has been described above, but the present invention is not limited to this.
[0075]
In the above embodiment, as the nucleic acid containing the nucleotide sequence encoding the bone morphogenetic protein (BMP), a recombinant plasmid in which BMP cDNA was incorporated into an expression plasmid was described as a representative example. The nucleic acid to be contained may be, for example, BMP cDNA (which is not incorporated into an expression plasmid), BMP mRNA, or a product obtained by adding an arbitrary base thereto.
[0076]
【Example】
Next, specific examples of the present invention will be described.
[0077]
(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.
Then, this recombinant plasmid was propagated as follows.
[0078]
First, at room temperature, the recombinant plasmid was added to 200 µL of a suspension of DH5α (Competent Bacteria).
[0079]
Next, this mixture was added to LB agar medium and cultured at 37 ° C. for 12 hours.
Next, after the completion of the culture, a relatively large colony was selected from the colonies grown on the LB agar medium, transplanted to an LB agar medium containing Amp (ampicillin), and further cultured at 37 ° C. for 12 hours. .
[0080]
Thereafter, the cell membrane of DH5α grown on the LB agar medium containing Amp was disrupted, and the recombinant plasmid was purified and separated from the solution.
[0081]
2. Manufacture of hydroxyapatite porous sintered body (substrate)
Hydroxyapatite was synthesized by a known wet synthesis method to obtain a hydroxyapatite slurry.
[0082]
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 a mean particle size of about 12 μm using a general-purpose pulverizer. The mixture containing the pulverized hydroxyapatite powder and the water-soluble polymer was stirred in order to foam the mixture to form a paste. The hydroxyapatite powder and the water-soluble polymer were mixed at a weight ratio of 5: 6.
[0083]
This paste-like kneaded product was put in a mold, and dried at 80 ° C. to gel the water-soluble polymer to produce 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.
[0084]
The disc-shaped compact was fired at 1200 ° C. in the air for 2 hours to obtain a hydroxyapatite porous sintered body.
[0085]
The porosity of the porous hydroxyapatite sintered body was 70%. This measurement was performed by the Archimedes method.
[0086]
3. Fabrication of osteogenic treatment device
A phosphate buffer solution containing the recombinant plasmid and a phosphate buffer solution containing a positively charged liposome (QIAGEN, “SuperFect”) as a vector were prepared. The porous hydroxyapatite sintered body was impregnated so as to be 40 μg.
As a result, an osteogenic treatment device (n = 6) was obtained.
[0087]
(Comparative example)
An osteogenic treatment device (n = 6) was prepared in the same manner as in the above example except that no recombinant plasmid was used.
[0088]
<Evaluation>
1. Evaluation experiment
Eighteen rabbits (average weight 3.0 kg) were prepared. The following operations were performed on each rabbit.
[0089]
First, rabbits were anesthetized by intravenously administering 25 mg / kg sodium pentobarbital (manufactured by Abbott Laboratories, "Nembutal").
[0090]
Next, an incision was made in the scalp of the rabbit, and the rabbit was raised as a flap having a width of 2.5 cm and a length of 3.0 cm with the stem on the tail side.
[0091]
Next, a 2-3 mm incision was made in the exposed periosteum, a periosteal exfoliator was applied to such a portion, and a portion having a diameter of about 3 mm was peeled to expose the skull.
[0092]
Next, the exposed median skull was opened near the median using a skull perforator, and the skull was removed immediately above the dura so as to preserve the dura, followed by complete hemostasis. The thickness of the skull was about 3 mm, and the diameter of the craniotomy was about 1.2 cm.
[0093]
Next, the rabbits subjected to craniotomy were divided into two groups of nine rabbits. The rabbits of the first group were treated with the osteogenesis treatment device of the example, and the rabbits of the second group were treated with the rabbits. After implanting the osteogenesis treatment device of the comparative example, the flap was returned to the original position and sutured.
[0094]
Then, each rabbit subjected to the operation was bred in an individual cage.
[0095]
2. Evaluation results
At 3, 6, and 9 weeks after the operation, three rabbits in each group were sacrificed by overdose of the same anesthetic as described above.
[0096]
Thereafter, the skull was excised as a lump together with the skin directly above, and the collected tissue was immediately immersed and fixed in 10% neutral buffered formalin solution, and then embedded in a polyester resin. Next, the tissue embedded in the polyester resin was sliced and polished to a thickness of 50 μm, and then subjected to cole-HE staining. Thus, a tissue specimen was obtained.
[0097]
The bone formation rate of each of the obtained tissue specimens was measured as follows. That is, each tissue specimen was photographed with a stereo microscope system SZX-12 (manufactured by Olympus Corporation) equipped 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, the extracted bone is extracted by an image analysis technique using a SCION image (manufactured by Scion). The area of the new bone portion was measured and quantified to determine the bone formation rate.
[0098]
In addition, the measurement of the bone formation rate was performed by measuring 5 mm × (thickness of the hydroxyapatite porous sintered body 3 mm + hardening side) from the end in the surface direction (direction perpendicular to the thickness direction) of the hydroxyapatite porous sintered body. 2 mm portion).
Table 1 shows the results.
[0099]
[Table 1]

[0100]
As shown in Table 1, the osteogenesis treatment devices of the examples were extremely excellent in osteogenic ability.
In contrast, the bone formation treatment device of the comparative example was inferior in bone formation ability.
[0101]
As described above, it has been clarified that a combined use of a porous hydroxyapatite sintered body, a recombinant plasmid, and a positively charged liposome results in a bone formation treatment device having extremely excellent bone formation ability.
[0102]
Further, instead of the hydroxyapatite porous sintered body as a substrate, a bone formation treatment device was prepared using a tricalcium phosphate porous sintered body, and the same evaluation experiment was performed as a result. Similar evaluation results were obtained.
[0103]
Also, as the base sequence encoding BMP, human BMP-1, human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP- 6, using a nucleotide sequence encoding human BMP-7, human BMP-8, human BMP-9 or human BMP-12 to produce an osteogenic therapeutic device, and performing the same evaluation experiment as described above, Almost the same evaluation results as in the example were obtained.
[0104]
【The invention's effect】
As described above, according to the present invention, extremely rapid bone formation can be achieved, and it is possible to contribute to early bone formation treatment.
[0105]
Therefore, it is not necessary to perform free bone transplantation in various osteogenesis treatments, and a bone collecting part is not required. Therefore, safer, more reliable and rational surgery can be performed.
[0106]
For this reason, it is possible to reduce the operation time and hospitalization period, to reduce the total medical cost, improve the quality of treatment, and improve the QOL of the patient.
[0107]
In particular, in the present invention, by using a vector in combination, the efficiency of incorporation of nucleic acids into cells involved in bone formation such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts is improved. The bone formation is performed more quickly, and the effect is further improved.
[0108]
In addition, by using a block body as the base, bone formation proceeds favorably along the shape of the base, which is effective when the transplant site is a relatively large bone defect or the like.
[0109]
In addition, by using a porous body specialized for gene therapy as a substrate, nucleic acids and vectors can be more easily and reliably supported on the substrate, and various cells involved in bone formation can be contained in the substrate. It is easier to penetrate and is advantageous for bone formation.
[0110]
Further, the osteogenic treatment device of the present invention is easy to store, handle, and process during surgery.
[Brief description of the drawings]
FIG. 1 is a genetic map showing an example of a recombinant plasmid.

Claims (12)

A nucleic acid comprising a base sequence encoding a bone morphogenetic protein (BMP);
A vector holding the nucleic acid,
An osteogenic treatment device comprising: a biocompatible substrate. The osteogenic treatment device according to claim 1, wherein the bone morphogenetic protein is BMP-2. The osteogenesis treatment device according to claim 1, wherein the nucleic acid includes a base sequence derived from an expression plasmid. The bone formation treatment device according to any one of claims 1 to 3, wherein the nucleic acid is used in an amount of 1 to 100 µg per 1 mL of the volume of the substrate. The osteogenesis treatment device according to any one of claims 1 to 4, wherein the vector is a non-virus-derived vector. The osteogenic treatment device according to claim 5, wherein the non-virus-derived vector is a liposome. The device according to claim 6, wherein the liposome is a positively charged liposome. The osteogenesis treatment device according to any one of claims 1 to 7, wherein a mixing ratio of the vector and the nucleic acid is 1: 1 to 20: 1 by weight. The osteogenesis treatment device according to any one of claims 1 to 8, wherein the base is a block. The bone formation treatment device according to any one of claims 1 to 9, wherein the base is a porous body. The bone formation treatment device according to claim 10, wherein the porosity of the porous body is 30 to 95%. The osteogenesis treatment device according to any one of claims 1 to 11, wherein the base is mainly composed of hydroxyapatite or tricalcium phosphate.

Images