JP2004187739A - Osteogenic treatment device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an osteogenic treatment device capable of controlling the pattern of osteogenesis. <P>SOLUTION: The osteogenic treatment device is provided with nucleic acid including a base sequence coding bone morphogenetic protein (BMP) which is a bone inducing factor and a base body 1 with biocompatibility constituted of a porous block body, and is transplanted inside a living body to perform osteogenic treatment. At the time of defining the area (average) of the boundary part of adjacent holes 1a of the base body 1 as A[μm<SP>2</SP>] and the maximum cross section area (average) of the hole 1a as B[μm<SP>2</SP>], the osteogenic treatment device controls the pattern of the osteogenesis corresponding to the size of the value of B/A. For instance, in the case that B/A is a value less than 50 and in the case that the B/A is the value of 50 or higher, the pattern of the osteogenesis is controlled. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an osteogenesis treatment device (osteogenesis control 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 the osteoinductive factor, a bone morphogenetic protein (Bone Morphogenetic 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 (substrate).
[0008]
This matrices material is usually formed corresponding to the shape of a transplant site such as a bone defect where the transplant material is transplanted. Then, at the implantation site, the matrix material becomes a site for bone formation.
[0009]
Here, in some cases, in order to achieve osteogenesis treatment at an early stage, it is preferable that osteogenesis inside the matrix material progresses preferentially, or osteogenesis near the outer surface of the matrix material. There are various cases where it is preferable to proceed with priority.
[0010]
However, it is difficult to control such a pattern of bone formation with conventional implant materials.
[0011]
[Patent Document 1]
Japanese Unexamined Patent Publication No. 2001-505097
[Problems to be solved by the invention]
An object of the present invention is to provide an osteogenic treatment device capable of controlling an osteogenesis pattern.
[0013]
[Means for Solving the Problems]
Such an object is achieved by the present invention described in the following (1) to (19).
[0014]
(1) An osteogenesis treatment device comprising: an osteoinductive factor; and a base made of a porous block having continuous pores in which adjacent pores communicate with each other,
When the area (average) of the boundary between adjacent holes in the substrate is A [μm ² ] and the maximum cross-sectional area (average) of the holes is B [μm ² ], the value of B / A An osteogenesis treatment device characterized in that a pattern of osteogenesis can be controlled according to the size.
Thereby, the pattern of bone formation can be controlled.
[0015]
(2) The bone formation treatment device according to (1), wherein two or more types of the bases having different values of the B / A are prepared, and these are selectively used.
This makes it possible to perform a bone formation treatment in accordance with the purpose.
[0016]
(3) In the first case where the value of B / A is less than 50, and in the second case where the value of B / A is 50 or more, the above (1) or (1) which can control the pattern of bone formation. The bone formation treatment device according to 2).
[0017]
Thus, for example, a bone formation pattern in which bone formation proceeds preferentially inside the base and a bone formation pattern in which bone formation proceeds preferentially on the outer surface of the base can be controlled.
[0018]
(4) The bone formation treatment device according to (3), wherein a difference between the B / A value in the first case and the B / A value in the second case is 30 or more.
[0019]
Thereby, the difference in the tendency of the bone formation pattern between the first case and the second case (according to the magnitude relationship between the values of B / A) can be further clarified.
[0020]
(5) The bone formation treatment device according to any of (1) to (4), wherein the osteoinductive factor is a nucleic acid containing a base sequence encoding a bone morphogenetic protein (BMP).
Thereby, more rapid bone formation can be promoted.
[0021]
(6) The bone formation treatment device according to (5), wherein the bone morphogenetic protein is at least one of BMP-2, BMP-4, and BMP-7.
[0022]
BMP-2, BMP-4, and BMP-7 are particularly excellent in inducing the differentiation of undifferentiated mesenchymal cells into osteoblasts.
[0023]
(7) The device for treating osteogenesis according to the above (5) or (6), wherein the nucleic acid contains a base sequence derived from an expression plasmid.
[0024]
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.
[0025]
(8) The device for treating osteogenesis according to any one of (5) to (7), 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.
[0026]
(9) The device for treating osteogenesis according to any one of the above (5) to (8), comprising a vector retaining the nucleic acid.
[0027]
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.
[0028]
(10) The device for treating osteogenesis according to (9), wherein the vector is a non-virus-derived vector.
[0029]
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.
[0030]
(11) The device for treating osteogenesis according to (10), wherein the non-virus-derived vector is a liposome.
[0031]
Since liposomes are composed of components close to those of the cell membrane, binding (fusion) to the cell membrane is relatively easy and smooth, and nucleic acids such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts The efficiency of uptake into cells involved in bone formation can be further improved.
[0032]
(12) The osteogenic treatment device according to (11), wherein the liposome is a positively charged liposome.
[0033]
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.
[0034]
(13) The osteogenesis treatment device according to any one of (9) to (12), wherein the mixing ratio of the vector and the nucleic acid is 1: 1 to 20: 1 by weight.
[0035]
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.
[0036]
(14) The osteogenesis treatment device according to any one of (1) to (13), further comprising an angiogenesis inducing factor.
This promotes faster bone formation.
[0037]
(15) The bone according to (14), wherein the angiogenesis-inducing factor is at least one of fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). Plastic therapy device.
[0038]
Since these devices are excellent in angiogenic ability, the resulting osteogenic treatment device has particularly high osteogenic ability.
[0039]
(16) The osteogenesis treatment device according to (14) or (15), wherein the compounding ratio of the angiogenesis-inducing factor and the osteoinductive factor is from 10: 1 to 1: 100 by weight.
[0040]
Thus, new blood vessels can be efficiently formed, and osteoblasts can be efficiently proliferated.
[0041]
(17) The bone formation treatment according to any one of (1) to (16), wherein the maximum cross-sectional area (average) B of the pore is 7.9 × 10 ^{3 to} 1.1 × 10 ⁶ μm ^2. device.
[0042]
This makes it possible to make the substrate a more suitable site for bone formation while suitably maintaining the mechanical strength of the substrate.
[0043]
(18) The bone formation treatment device according to any one of (1) to (17), wherein the porosity of the base is 30 to 95%.
[0044]
This makes it possible to make the substrate a more suitable site for bone formation while suitably maintaining the mechanical strength of the substrate.
[0045]
(19) The bone formation treatment device according to any one of (1) to (18), wherein the base is mainly composed of hydroxyapatite or tricalcium phosphate.
[0046]
Hydroxyapatite and tricalcium phosphate have particularly excellent biocompatibility because they have the same (approximate) composition, structure and physical properties as the inorganic main components of bone.
[0047]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the osteogenesis treatment device (osteogenesis control device) of the present invention will be described in detail.
[0048]
The osteogenesis treatment device of the present invention includes an osteoinductive factor and a substrate, and is implanted in a living body to perform osteogenesis treatment.
[0049]
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.
[0050]
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.
[0051]
In the following description, a nucleic acid containing a base sequence encoding a bone morphogenetic protein (BMP) will be described as a typical osteoinductive factor. Use of a nucleic acid containing a base sequence encoding BMP as an osteoinductive factor can promote more rapid bone formation.
[0052]
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”.
[0053]
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 these, BMP is particularly preferably at least one of BMP-2, BMP-4 and BMP-7. Since BMP-2, BMP-4, and BMP-7 are particularly excellent in inducing the differentiation of undifferentiated mesenchymal cells into osteoblasts, the resulting osteogenic treatment device has a particularly high osteogenic ability. Show.
[0054]
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, as the BMP cDNA, the same as the nucleotide sequence encoding a naturally occurring BMP, or the nucleotide sequence in which one or more nucleotides are deleted, substituted and / or added in the nucleotide sequence encoding a naturally occurring BMP is used. Can be used. These may be used alone or in combination of two or more.
[0055]
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.
[0056]
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.
[0057]
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.
[0058]
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.
[0059]
The expression plasmid can be selected from those widely used in the field of genetic recombination engineering technology. For example, one or a combination of two or more of pCAH, pSC101, pBR322, and pUC18 can be used.
[0060]
In addition, a base sequence (DNA fragment) that appropriately controls BMP expression can be appropriately introduced into this recombinant plasmid.
[0061]
Known methods can be used for incorporating various base sequences including BMP cDNA into the expression plasmid.
[0062]
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.
[0063]
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.
[0064]
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.
[0065]
Further, the osteogenesis treatment device of the present invention has a base made of a porous block (for example, a sintered body). This substrate serves as a field for bone formation by osteoblasts differentiated from undifferentiated mesenchymal cells.
[0066]
This porous block body has continuous pores communicating with each other, instead of closed pores in which adjacent pores are closed.
[0067]
FIG. 2 is a schematic view showing a vertical cross section of the base according to the present invention, and FIGS. 3 and 4 are schematic views each showing an example of a bone formation pattern. The symbol “2” in FIGS. 3 and 4 indicates a new bone.
[0068]
In view of the above problems, the present inventor has conducted extensive studies, and as a result, has found that by appropriately setting the internal shape (void shape) of the base, the pattern of bone formation is different.
[0069]
Further, as a result of the research, as shown in FIG. 2, the present inventor sets the area (average) of the boundary portion between adjacent holes 1a of the substrate 1 to A [μm ² ], and Assuming that the maximum cross-sectional area (average) is B [μm ² ], it has been found that the bone formation pattern as described above largely depends on the value of B / A.
[0070]
The present invention has been made based on these findings. That is, the present invention is characterized in that a bone formation pattern can be controlled according to the magnitude of the value of B / A.
[0071]
For example, in the first case where the value of B / A is less than 50 and in the second case where the value of B / A is 50 or more, the pattern of bone formation shows the following tendency.
[0072]
Specifically, when the value of B / A is less than 50, in other words, the area (average) A of the boundary between the adjacent holes 1a is larger than the maximum cross-sectional area (average) B of the holes 1a. When the size is relatively large, the new bone 2 tends to be preferentially formed inside the base 1 as shown in FIG. It is presumed that this is because, when A is large, cells involved in bone formation are smoothly moved between adjacent holes 1a.
[0073]
In this case, the cells involved in bone formation move (diffuse) so as to deviate (leave) after sufficiently filling the pores 1a, and as shown in FIG. It will be formed corresponding to the shape of the base 1.
[0074]
On the other hand, when the value of B / A is 50 or more, in other words, the area (average) A of the boundary between the adjacent holes 1a is relatively larger than the maximum cross-sectional area (average) B of the holes 1a. When small, the new bone 2 tends to be formed not only inside the base 1 but also around its outer surface 1b (particularly, the outer surface 1b vertically below the base 1) as shown in FIG. This is presumed to be due to the fact that cells involved in osteogenesis infiltrate into the inside of the substrate 1 due to the small A.
[0075]
Such a value of B / A between the first case and the second case is preferably as good as possible, and the difference is preferably 30 or more, and more preferably 40 or more. Thereby, the difference in the tendency of the bone formation pattern between the first case and the second case (according to the magnitude relationship between the values of B / A) can be further clarified.
[0076]
Specifically, the value of B / A in the first case is preferably about 2 to 6, and the value of B / A in the second case is preferably about 50 to 150. Thereby, the tendency of the pattern of bone formation becomes even more remarkable, and the difference in the tendency of the pattern of bone formation between the first case and the second case (according to the magnitude relationship of the value of B / A) is determined. Can be particularly clear.
[0077]
For this reason, according to the present invention, two or more types of substrates 1 having different B / A values are prepared according to the purpose (for example, case, treatment site, treatment purpose, etc.), and these are selectively used. In this case, a suitable bone formation treatment, that is, an early bone formation treatment can be performed. Thereby, the QOL of the patient can be improved.
[0078]
The maximum cross-sectional area (average) B of the pore is preferably about 7.9 × 10 ^{3 to} 1.1 × 10 ⁶ μm ² , and 1.8 × 10 ^{4 to} 7.9 × 10 ⁵ μm ^2. More preferably, it is about When the maximum cross-sectional area (average) B of the pores is converted into an average pore diameter, the value is 100 to 1200 μm (preferably 150 to 1000 μm). By setting the maximum cross-sectional area (average) B of the pores in the above range, it is possible to maintain the mechanical strength of the base suitably, and to make the base a more suitable site for bone formation.
[0079]
Further, the porosity of the substrate 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, the base can be a more suitable site for bone formation while suitably maintaining the mechanical strength of the base.
[0080]
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.
[0081]
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.
[0082]
Hydroxyapatite and tricalcium phosphate have particularly excellent biocompatibility because they have the same (approximate) composition, structure, and properties as the inorganic main components of bone. In addition, since the liposome has both positive and negative charges, particularly when a liposome is used as a vector (this point will be described in detail later), 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.
[0083]
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 substrate will be described. For example, such a porous block is dried by stirring and foaming a ceramic slurry containing a water-soluble polymer. It can be manufactured by obtaining a molded body in a shape corresponding to a transplantation site such as a bone defect part by a general-purpose processing machine such as a machining center, and sintering (firing) the molded body.
[0084]
In such a method for producing a substrate, for example, conditions for synthesizing the raw material powder (primary particle size, primary particle dispersion state, etc.), conditions for the raw material powder (average particle size, presence or absence of calcination, presence or absence of pulverization treatment) Etc.), the conditions of the stirring and foaming (type of surfactant, stirring power for stirring the slurry, etc.), firing conditions (firing atmosphere, firing temperature, etc.), etc. are appropriately set, so that the value of B / A is desired. Can be set to For example, when the firing temperature is increased, diffusion between the raw material powders is promoted, and the value of A tends to decrease and the value of B / A tends to increase.
[0085]
In particular, since the conditions of the raw material powder and the conditions of stirring and foaming greatly affect the value of B / A, it is preferable to control these conditions more strictly.
[0086]
Note that various conditions for obtaining the target value of B / A can be experimentally obtained.
[0087]
The bone formation treatment device as described above can be produced (manufactured) by bringing a recombinant plasmid (nucleic acid) into contact with a substrate. Specifically, the osteogenic treatment device is easily manufactured by, for example, supplying a liquid (solution or suspension) containing the recombinant plasmid to the substrate, or immersing the substrate in the liquid. be able to.
[0088]
Further, for example, an osteogenesis treatment device can be produced by molding a kneaded product obtained by kneading a powdery, granular, or pellet-like precursor of a substrate, a binder, and a liquid as described above.
[0089]
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. In addition, this bone formation progresses rapidly with different bone formation patterns depending on the magnitude of the B / A value.
[0090]
Further, such an osteogenic treatment device preferably contains a vector and an angiogenesis-inducing factor. Inclusion of these in the osteogenic treatment device promotes more rapid bone formation. The osteogenesis treatment device preferably includes either one, and more preferably includes both.
[0091]
Hereinafter, the vector and the angiogenesis-inducing factor will be described in detail.
[0092]
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.
[0093]
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.
[0094]
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.
[0095]
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.
[0096]
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.
[0097]
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.
[0098]
In addition, an additive for stabilizing a lipid membrane such as cholesterol may be added to these liposomes.
[0099]
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.
[0100]
The amount of the vector to be used is appropriately set depending on the type and the like, and is not particularly limited. The mixing ratio between 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 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.
[0101]
An angiogenesis-inducing factor acts on cells involved in angiogenesis (eg, vascular skin cells) to promote the formation of new blood vessels. By using this angiogenesis-inducing factor, new blood vessels are formed around osteoblasts. As a result, various substrates necessary for cell construction (formation) are supplied to the osteoblasts, so that the osteoblasts can proliferate efficiently. As a result, bone formation can be further promoted.
[0102]
The angiogenesis-inducing factor in the present invention is not particularly limited as long as it can promote angiogenesis, and examples thereof include, but are not limited to, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (bFGF). Vascular Endothelial Growth Factor: VEGF, Hepatocyte Growth Factor (HGF), Granulocyte Macrophage Colony Stimulating Gastrointegrative Factor-Granocyte Cytometry G-CSF), macrophage colony stimulating factor (Ma Rophage-Colony Stimulating Factor: M-CSF, Stem Cell Factor (Stem Cell Factor: SCF), Angiopoietin-1, Angiopoietin-2, Angiopoietin amide, protein analog E (prostaglandin E ₁ , prostaglandin E ₂ , prostaglandin E ₃ ), proline derivatives, cyclic AMP derivatives such as dibutyl cyclic AMP (dBcAMP), and one of these. Alternatively, two or more kinds 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 devices are excellent in angiogenic ability, the resulting osteogenic treatment device has particularly high osteogenic ability.
[0103]
The amount of the angiogenesis-inducing factor to be used is appropriately set depending on the type and the like, and is not particularly limited. And more preferably about 1: 1 to 1: 100. If the amount of the angiogenesis-inducing factor used is too small, new blood vessels may not be efficiently formed depending on the type of the angiogenesis-inducing factor or the like, and osteoblasts may not be sufficiently proliferated. On the other hand, even if the amount of the angiogenesis-inducing factor used exceeds the upper limit, no further increase in the effect can be expected.
[0104]
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.
[0105]
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.
[0106]
Further, as the osteoinductive factor, a nucleic acid containing a base sequence encoding a bone morphogenetic protein (BMP) has been described as a representative, but the osteoinductive factor in the present invention includes the above-mentioned BMP itself and the like.
[0107]
【Example】
Next, specific examples of the present invention will be described.
[0108]
(Example 1)
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, and the recombinant plasmid as shown in FIG. Got.
Then, this recombinant plasmid was propagated as follows.
[0109]
First, at room temperature, the recombinant plasmid was added to 200 µL of a suspension of DH5α (Competent Bacteria).
Next, this mixture was added to LB agar medium and cultured at 37 ° C. for 12 hours.
[0110]
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. .
[0111]
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.
[0112]
2. Production of hydroxyapatite porous sintered body (substrate) Hydroxyapatite was synthesized by a known wet synthesis method to obtain a hydroxyapatite slurry.
[0113]
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 pulverized hydroxyapatite powder was mixed with a 1% aqueous solution of methylcellulose (water-soluble polymer) and then stirred to obtain a paste-like mixture containing bubbles. The hydroxyapatite powder and the aqueous methylcellulose solution were mixed at a weight ratio of 5: 6.
[0114]
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 formed body was processed into a disk 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.
[0115]
The disc-shaped compact was fired at 1200 ° C. in the air for 2 hours to obtain a hydroxyapatite porous sintered body.
[0116]
The porosity of the porous hydroxyapatite sintered body was 70%. This measurement was performed by the Archimedes method. The B / A was about 100.
[0117]
3. Preparation of a device for treatment of osteogenesis A phosphate buffer containing a recombinant plasmid, a phosphate buffer containing a basic fibroblast growth factor (bFGF) which is an angiogenesis inducing factor, and a positively charged liposome (a vector) A phosphate buffer solution containing “SuperFect” manufactured by QIAGEN Co., Ltd.) was prepared. The porous sintered body was impregnated.
Thus, an osteogenic treatment device was obtained.
[0118]
(Example 2)
The same hydroxyapatite powder as in Example 1 and N, N-dimethyldodecylamine oxide (“AROMOX” manufactured by Lion Corporation) as a nonionic surfactant were mixed with 1% methylcellulose (water-soluble (Molecule) After mixing with the aqueous solution, the mixture was vigorously stirred as in Example 1 to obtain a paste-like mixture containing bubbles. Note that N, N-dimethyldodecylamine oxide was added to the paste-like mixture so as to be 2 wt%.
[0119]
An osteogenic treatment device was produced in the same manner as in Example 1 except that a porous hydroxyapatite sintered body (substrate) was produced using the paste-like mixture.
[0120]
The hydroxyapatite porous sintered body (substrate) had a B / A of about 3 and a porosity of 85%.
[0121]
<Evaluation>
1. Evaluation Experiment Twelve rabbits (average body weight: 3.0 kg) were prepared. The following operations were performed on each rabbit.
[0122]
First, rabbits were anesthetized by intravenously administering 25 mg / kg sodium pentobarbital (manufactured by Abbott Laboratories, "Nembutal").
[0123]
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.
[0124]
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.
[0125]
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.
[0126]
Next, the head-opened rabbits were divided into a total of two groups of six rabbits, and each of the rabbits of the first group was applied with the osteogenesis treatment device of Example 1 to each of the rabbits of the second group. After implanting the osteogenic treatment device of Example 2, the flaps were returned to their original positions and sutured.
[0127]
Then, each rabbit subjected to the operation was bred in an individual cage.
[0128]
2. Evaluation results Nine weeks after the operation, all rabbits were sacrificed by overdose of the same anesthetic as described above.
[0129]
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.
[0130]
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).
[0131]
As a result, a similar bone formation pattern was observed in each of the six birds in the first group, and a similar bone formation pattern was observed in each of the six birds in the second group.
[0132]
FIG. 5 shows an example of an observation image (image) of the first group of tissue specimens, and FIG. 6 shows an example of an observation image (image) of the second group of tissue specimens.
[0133]
As shown in these figures, in the first group, that is, in the rabbit to which the osteogenesis treatment device of Example 1 was implanted, the bone formation was caused not only inside the porous hydroxyapatite sintered body but also vertically below the porous hydroxyapatite sintered body. In the second group, ie, rabbits transplanted with the osteogenesis treatment device of Example 2, bone formation preferentially progresses inside the osteogenesis treatment device. Showed a trend.
[0134]
Thus, it has become clear that the pattern of bone formation can be controlled by appropriately setting B / A.
[0135]
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.
[0136]
Further, as an osteoinductive factor, various human BMPs, nucleic acids containing nucleotide sequences encoding various human BMPs, drugs such as prostaglandins, or any combination thereof to produce an osteogenic treatment device, and the same as above As a result of the evaluation experiment, almost the same evaluation results as in the above example were obtained.
[0137]
In addition, as an angiogenesis-inducing factor, instead of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) or hepatocyte growth factor (HGF), or any combination thereof, to provide an osteogenesis treatment device. As a result of performing the same evaluation experiment as described above, almost the same evaluation results as in the above-described example were obtained.
[0138]
【The invention's effect】
As described above, according to the present invention, a pattern of bone formation can be controlled.
[0139]
Therefore, by controlling the pattern of osteogenesis according to the purpose, osteogenesis treatment suitable for each, that is, early osteogenesis treatment can be performed.
[0140]
Further, according to the present invention, it is not necessary to perform a free bone transplantation in various osteogenesis treatments, and a bone collecting part is not required, so that safer, more reliable and rational surgery can be performed.
[0141]
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.
[0142]
In addition, by using the angiogenesis-inducing factor together, new blood vessels are actively formed around the osteoblasts, and the osteoblasts are efficiently proliferated. As a result, more rapid bone formation is achieved.
[0143]
In addition, by using a vector holding a nucleic acid in combination, the efficiency of uptake of the nucleic acid into cells involved in bone formation such as undifferentiated mesenchymal cells, inflammatory cells, and fibroblasts is improved. More rapid bone formation.
[0144]
Further, by controlling the value of B / A of the substrate, for example, various osteoinductive factors, various vectors, various angiogenesis inducing factors, etc. can be added to the optimal site of the substrate according to the purpose and in the optimal amount. Can be carried (held). From such a point, the osteogenesis treatment device of the present invention enables osteogenesis treatment suitable for the purpose, that is, early osteogenesis treatment.
[0145]
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.
FIG. 2 is a schematic diagram showing a vertical cross section of a base in the present invention.
FIG. 3 is a schematic diagram showing an example of a bone formation pattern.
FIG. 4 is a schematic diagram showing an example of a bone formation pattern.
FIG. 5 is an image showing an example of an observation image of a tissue specimen of the first group.
FIG. 6 is an image showing an example of an observation image of a tissue specimen of the second group.
[Explanation of symbols]
1 Base 1a Void 1b Outer surface 2 New bone

Claims (19)

An osteoinductive treatment device comprising an osteoinductive factor and a base formed of a porous block having continuous pores in which adjacent pores communicate with each other,
When the area (average) of the boundary between adjacent holes in the substrate is A [μm ² ] and the maximum cross-sectional area (average) of the holes is B [μm ² ], the value of B / A An osteogenesis treatment device characterized in that a pattern of osteogenesis can be controlled according to the size. The osteogenesis treatment device according to claim 1, wherein two or more types of bases having different values of B / A are prepared, and these are selectively used. The bone according to claim 1, wherein a bone formation pattern can be controlled in a first case where the B / A value is less than 50 and in a second case where the B / A value is 50 or more. 4. Plastic therapy device. The osteogenesis treatment device according to claim 3, wherein a difference between the B / A value in the first case and the B / A value in the second case is 30 or more. The osteogenesis treatment device according to any one of claims 1 to 4, wherein the osteoinductive factor is a nucleic acid containing a base sequence encoding a bone morphogenetic protein (BMP). The device according to claim 5, wherein the bone morphogenetic protein is at least one of BMP-2, BMP-4, and BMP-7. The osteogenesis treatment device according to claim 5, wherein the nucleic acid includes a base sequence derived from an expression plasmid. The device according to any one of claims 5 to 7, 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 5 to 8, comprising a vector holding the nucleic acid. The osteogenic treatment device according to claim 9, wherein the vector is a non-virus-derived vector. The osteogenic treatment device according to claim 10, wherein the non-virus-derived vector is a liposome. The device according to claim 11, wherein the liposome is a positively charged liposome. The bone formation treatment device according to any one of claims 9 to 12, wherein a mixing ratio of the vector and the nucleic acid is 1: 1 to 20: 1 by weight. The bone formation treatment device according to any one of claims 1 to 13, further comprising an angiogenesis-inducing factor. The osteogenic treatment device according to claim 14, wherein the angiogenesis-inducing factor is at least one of fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). The osteogenesis treatment device according to claim 14 or 15, wherein a compounding ratio of the angiogenesis-inducing factor and the osteoinductive factor is from 10: 1 to 1: 100 by weight. The maximum cross-sectional area of the holes (average) B is, 7.9 × ¹⁰ 3 ~1.1 claims 1 a × 10 ⁶ μm ² to 16 osteogenic treatment device according to any one of. The osteogenesis treatment device according to any one of claims 1 to 17, wherein the porosity of the base is 30 to 95%. 19. The bone formation treatment device according to claim 1, wherein the base is mainly composed of hydroxyapatite or tricalcium phosphate.

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