JP2004254751A - Biological tissue prosthesis material, production method of biological tissue prosthesis, container used therefor, and biological tissue prosthesis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biological tissue prosthesis material, a production method of the biological prosthesis and a container used therefor which enable an even and sufficient remodelling by growing the cells without fault even when a relatively large defect part is filled. <P>SOLUTION: A block-shaped biological tissue prosthesis material 1 has a through-hole 2 and a fluid restriction part 2b disposed at the through-hole 2 to restrict the stream of the cells flowing therein. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a living tissue filling material, a method for producing a living tissue filling, and a container used for the same.
[0002]
[Prior art]
2. Description of the Related Art In recent years, it has become possible to regenerate bone and repair the defect by replenishing a bone replacement material into a defect in the bone caused by removal of a bone tumor or trauma. Hydroxyapatite (HAP) and tricalcium phosphate (TCP) are known as bone replacement materials. However, from the viewpoint that foreign substances are not left in the body, for example, a calcium phosphate porous material such as β-TCP is used. Scaffolding is used. When β-TCP is brought into contact with bone cells in a bone defect, so-called remodeling is performed in which osteoclasts eat β-TCP and osteoblasts form new bone. That is, the bone replacement material that has been repaired in the bone defect part is replaced with autologous bone over time.
[0003]
On the other hand, it has been proposed to use cultured bone produced by culturing bone marrow mesenchymal cells collected from a patient together with a bone filling material in order to increase the repair speed of a bone defect after surgery. The cultured bone containing many bone marrow mesenchymal cells that grew using the bone filling material as a scaffold by culturing is filled in the bone defect, so that compared to the method of growing cells in the body after surgery, it becomes more autologous. The number of days until replacement is significantly reduced (for example, see Non-Patent Document 1).
[0004]
Such cultured bone is generally obtained by primary culture of bone marrow cells collected from a patient in a flask to increase the required number of cells, and then exfoliating the cells from the flask using a protease such as trypsin. Then, it is produced by attaching it to a bone filling material, placing it in an osteogenic medium, and performing secondary culture (for example, see Non-Patent Document 2).
[0005]
[Non-patent document 1]
Uemura et al., “Tissue Engineering in Bone Using Biodegradable β-TCP Porous Material-Ospherion, a New Material that Increases Strength in the Living Body”, Medical Asahi, Asahi Shimbun, October 1, 2001, No. 30 Vol. 10, No. 10, p. 46-49
[Non-patent document 2]
Yoshikawa, "Cultured dermis and cultured bone using bone marrow mesenchymal cells-regenerative medicine using bone marrow mesenchymal cells-", Bioindustry, CMC Publishing Co., Ltd., 2001, Vol. 18, No. 7, p. 46-53
[0006]
[Problems to be solved by the invention]
The biological tissue replacement material such as a bone replacement material is generally formed in a cubic block shape, and a material having a size suitable for the size of a defect portion of the biological tissue to be repaired is selected. Therefore, especially when the defect to be filled is large, a relatively large block-shaped living tissue filling material is used.
However, when the living tissue filling material becomes large, the cells seeded on the outer surface do not penetrate to the inside, and as a result, even if the cells are filled in the defective part of the living tissue, there may be a disadvantage that sufficient remodeling is not performed.
[0007]
The present invention has been made in view of the above-mentioned circumstances, and even when a relatively large defect is filled, a living tissue filling material capable of growing cells healthy and performing uniform and sufficient remodeling. It is an object of the present invention to provide a method for producing a body tissue complement and a container used for the method.
[0008]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides the following means.
The invention according to claim 1 is a block-shaped living tissue replenishing material, comprising a through-hole, wherein the through-hole is provided with a flow restricting portion for restricting a flow of cells to be flowed. I will provide a.
[0009]
According to the present invention, when the liquid containing cells is dropped on the living tissue filling material having the through hole, the liquid flows down from the surface of the living tissue filling material through the through hole. In this case, since the flow restricting portion is provided in the through hole, the flow of the cells is restricted, and the cell stays in the through hole. As a result, the seeded cells do not flow out through the through-holes, but are fixed inside the living tissue filling material.
[0010]
According to a second aspect of the present invention, there is provided the living tissue replacement material according to the first aspect, wherein the flow restricting portion is constituted by a small diameter portion in which a diameter of a through hole is partially reduced. .
According to the present invention, the flow restricting portion can be easily constituted by the small diameter portion. That is, the cells that have flowed into the through-hole are prevented from flowing further by the small diameter portion. In addition, the liquid other than the cells flows out through the small diameter portion. Therefore, the liquid does not accumulate in the through-hole and the inflow of cells into the through-hole is not hindered, so that the cells can be seeded efficiently.
[0011]
The invention according to claim 3 is a state in which the block-shaped living tissue replacement material having a through-hole is arranged so that the through-hole extends along the up-down direction, and the lower end of the through-hole is incompletely closed by a plug, Provided is a method for producing a biological tissue replacement in which a liquid containing cells flows down from above a through hole.
According to the present invention, by incompletely closing the lower end of the through hole with the stopper, it is possible to allow the flow of liquid other than cells while restricting the flow of cells. As a result, the seeded cells can be kept inside the living tissue filling material, and the living tissue filling material can be manufactured efficiently.
[0012]
According to a fifth aspect of the present invention, there is provided a container for use in the method for producing a living tissue replacement according to the third aspect, wherein the projection is provided on a bottom surface at a position corresponding to a through hole formed in the living tissue replacement material. Wherein the diameter of the projection is smaller than the diameter of the through hole at the upper end and larger than the diameter of the through hole at the lower end.
[0013]
According to the present invention, the lower end of the through hole is closed by the projection by placing the living tissue replacement material on the container such that the through hole matches the projection provided on the container. Since the projection has an upper end diameter smaller than the diameter of the through hole, the upper end is inserted into the through hole. On the other hand, since the projection has a lower end diameter larger than the diameter of the through hole, the projection is inserted into the through hole up to a middle position in the length direction. Thereby, a gap is formed between the lower surface of the living tissue replacement material placed on the protrusion and the bottom surface of the container, and the liquid flowing out between the through hole and the protrusion can flow through the gap. .
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
A living tissue replacement according to a first embodiment of the present invention will be described below with reference to FIG.
The living tissue filling material 1 according to the present embodiment is a filling material for filling a defect formed in a living tissue such as a bone, for example, and is a rectangular parallelepiped or a β-TCP porous material having a porosity of about 70%. It is configured as a cubic block. The living tissue filling material 1 is provided with a through-hole 2 penetrating from one surface 1a to the opposite surface 1b.
[0015]
The through-hole 2 is a stepped through-hole 2 having a large-diameter portion 2a having a large diameter and a small-diameter portion 2b (flow restriction portion) having a smaller diameter than the large-diameter portion 2a. The step between the large-diameter portion 2a and the small-diameter portion 2b is arranged at a substantially central position of the living tissue filling material 1.
[0016]
A case where a living tissue replacement is manufactured using the living tissue replacement 1 according to the present embodiment configured as described above will be described below.
As shown in FIG. 2, the living tissue replacement 1 is placed in an appropriate container 3 with the through-holes 2 arranged vertically with the small-diameter portion 2 b on the lower side and the large-diameter portion 2 a on the upper side. . Then, a liquid containing cells, for example, a mesenchymal stem cell (hereinafter, also referred to as a cell-containing liquid A) is dropped from above the living tissue filling material 1. Reference numeral 4 indicates, for example, an electric pipette.
[0017]
The cell-containing liquid A is dropped on the upper surface 1a of the living tissue replenishing material 1 to cause the contained cells to adhere to the surface of the living tissue replenishing material 1. When the cell-containing liquid A is dropped into the through-hole 2, the cells in the cell-containing liquid A adhere to the inner wall of the through-hole 2 while descending along the through-hole 2.
[0018]
In this case, in the biological tissue replenishing material 1 according to the present embodiment, since the step is provided in the through-hole 2, the cell-containing liquid A flowing into the through-hole 2 flows through the through-hole 2 as shown in FIG. 2 and is once blocked at the step without passing through. That is, the flow of the cell-containing liquid A inside the living tissue replacement material 1 is restricted by the step.
And, by restricting the flow of the cell-containing liquid A, the cells can be easily attached to the inner wall of the through-hole 2.
[0019]
In addition, the liquid other than the cells is caused to flow out through the small diameter portion 2b. Therefore, the restriction of inflow due to the accumulation of the cell-containing liquid A in the through-hole 2 is prevented, and it becomes possible to cause new cells to sequentially flow into the through-hole 2.
[0020]
In this way, according to the living tissue replenishing material 1 according to the present embodiment, the cell-containing liquid A is temporarily retained in the through-hole 2, so that the attachment of cells to the inner wall of the through-hole 2 can be promoted. Therefore, it is possible to efficiently manufacture the body tissue complement. Also, when culturing the living tissue supplement 1 to which cells are attached, the culture solution can be flowed into the through-holes 2 and waste products from the cells can be discharged from the through-holes 2. At this time, the step provided in the through hole 2 prevents the cells from flowing out of the through hole 2 due to the flow of the culture solution.
[0021]
Therefore, according to the living tissue filling material 1 according to the present embodiment, even when culturing the adhered cells, sufficient nutrient supply to the cells and waste products from the cells are prevented while preventing the cells from flowing out. Can be smoothly excreted to promote cell growth.
[0022]
In addition, in the biological tissue replenishing material 1 according to the present embodiment, the material having the stepped through hole 2 including the large diameter portion 2a and the small diameter portion 2b is illustrated, but is shown in FIG. 4 instead. As described above, the through-hole 2 having the hollow portion 2c having a larger diameter than the large-diameter portion 2a may be provided at the center.
Also, depending on the size of the living tissue filler 1, the cells may not sufficiently enter the inside of the living tissue filler 1, so if the large diameter portion 2a is smaller than the upper surface 1a, a plurality of through holes 2 are required. It may be provided in the living tissue filling material 1.
This makes it easier for the inflowing cells to stay in the living tissue replenishing material 1 and enhances the cell adhesion.
[0023]
As described above, when the hollow portion 2c having a large diameter is provided inside the living tissue replenishing material 1, it is difficult to process from the outside. Therefore, as shown in FIG. After being divided into two parts and processed separately, they may be combined and adhered with an arbitrary adhesive or the like.
Further, the outer shape of the living tissue replacement material 1 is not limited to a cubic shape, but may be a columnar shape such as a column. Further, as long as the through hole 2 has the large diameter portion 2a and the small diameter portion 2b, the shape of the through hole 2 as shown in FIG. 6 may be used.
[0024]
Next, a container for producing a biological tissue complement according to an embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 7, the manufacturing container 10 according to the present embodiment is formed in a petri dish shape capable of storing a culture medium and the like, and includes a plurality of protrusions 11 on the inner surface of the bottom 10a.
As shown in FIG. 8, these projections 11 are each formed in a substantially hemispherical shape, and are arranged at a predetermined pitch p.
[0025]
The pitch p of the protrusion 11 is formed in accordance with the living tissue replacement 12 to be used. That is, in order to manufacture a living tissue replacement using the manufacturing container 10 according to the present embodiment, a block-shaped living tissue replacement 12 having a plurality of through holes 13 is prepared as shown in FIG. Each through hole 13 has the same constant circular cross section, and is formed in parallel with the pitch p of the protrusion 11.
[0026]
The radius of the projection 11 is formed slightly larger than the radius of the through hole 13. As a result, as shown in FIG. 8, when the living tissue replacement material 12 is placed on the container 10 with the through hole 13 aligned with the protrusion 11, the protrusion 11 In this state, the living tissue replacement 12 is slightly lifted from the bottom surface 10 a of the container 10. At this time, each projection 11 functions as a plug that substantially closes the lower opening 13a of the through-hole 13. However, since the living tissue filler 12 is formed of a porous body, the lower opening 13a is not completely closed. It has become.
[0027]
The operation of the thus-configured manufacturing container 10 according to the present embodiment will be described below.
In order to manufacture a living tissue replacement using the manufacturing container 10 according to the present embodiment, as described above, the living tissue replacement 12 having a plurality of through holes 13 is held on the projection 11 of the manufacturing container 10. In this state, a cell-containing solution A such as mesenchymal stem cells is dropped from above.
[0028]
The dropped cell-containing liquid A adheres to the outer surface of the living tissue filling material 12 and flows into the plurality of through holes 13 to descend. In this case, according to the manufacturing container 10 according to the present embodiment, since the lower opening 13a of the through hole 13 provided in the living tissue replenishing material 12 is substantially closed by the protrusion 11, the cells flowing into the through hole 13 are formed. The contained liquid A is temporarily retained in the through hole 13. Thereby, the cells in the cell-containing liquid A stay in the through-hole 13, so that the cells easily adhere to the inner wall of the through-hole 13 and are seeded efficiently.
[0029]
Further, since a minute gap is formed between the lower opening 13a of the through hole 13 provided in the biological tissue filler 12 made of a porous body and the projection 11 for closing the same, the inside of the through hole 13 is formed. The liquid other than the inner cells of the cell-containing liquid A that has flowed in leaks out of the through-hole 13 through the gap. Therefore, the cells in the cell-containing liquid A are captured in the through holes 13.
[0030]
In addition, since the living tissue replenishing material 12 is held in a state of being slightly lifted from the bottom surface 10a of the container 10 by the projection 11, the flow of the liquid leaking from the through hole 13 is not hindered. Therefore, a new cell-containing liquid A can be sequentially dropped into the through-hole 13.
Further, in the living tissue replenishing material 12 to which the cells have been attached in this manner, the cells are cultured by storing a predetermined medium in the manufacturing container 10 and distributing the cells under predetermined culture conditions. . As a result, cells attached not only to the outer surface of the living tissue replacement material 12 but also to the through holes 13 grow using the living tissue replacement material 12 as a scaffold, and a living tissue replacement body having sufficiently grown cells inside is produced. Will be done.
Further, since the living tissue filler 12 can be placed in the culture medium with the living tissue filler 12 slightly floating from the bottom surface 10a of the container 10, cells can be grown on the lower surface of the biological tissue filler 12 as well.
[0031]
In this way, the block-shaped living tissue replenishing material 12 having the through-holes 13 is arranged so that the through-holes 13 extend in the up-down direction, and the lower end of the through-holes 13 is separated by a plug such as the projection 11 so as to be transparent. According to the method for producing a living tissue replacement body in which the liquid A containing cells flows down from above the through hole 13 in a state where the stopper is incompletely closed, the stepped living tissue replacement material 1 according to the above embodiment is used. In the same manner as in the above case, efficient seeding and growth of cells can be achieved.
[0032]
In the manufacturing container 10 according to the embodiment, the plurality of hemispherical projections 11 are arranged on the bottom surface 10 a of the container 10, but instead, the upper traverse is slightly smaller than the lower opening 13 a of the through hole 13. As long as the shape has a surface and a slightly larger lower cross section, for example, as shown in FIG. 10, a conical or frusto-conical projection 11 having a cone shape may be employed.
[0033]
In addition, as shown in FIG. 11, when a container 14 having a flat bottom such as a commercially available petri dish is used instead of the special container 10, the hole diameter of the living tissue replenishing material 15 near the lower end of the through hole 13 is changed. By providing a narrowed portion 15a to make the cell smaller, the cells stay in the through-hole 13, and by utilizing a gap A formed to some extent between the bottom surface of the container 14 and the bottom surface of the living tissue replenishing material 15, the flow of the culture solution can be reduced. Waste cells may be exchanged from the cells. In this case, the pitches p1 and p2 of the through holes 13 provided in the living tissue filler 15 may be different.
[0034]
In addition, as the living tissue replacement materials 1, 12, and 15, a material made of a β-TCP porous material has been described as an example. Adopt polymer materials, synthetic polymer materials such as polylactic acid and ε-caprolactan, metal materials and metal fibers such as titanium alloy, tantalum alloy and stainless steel 316, and composite materials of at least two or more of these materials May be.
[0035]
In addition, as the cells to be engrafted in the living tissue supplements 1, 12, and 15, other than mesenchymal stem cells contained in bone marrow cells, mesenchymal stem cells separated from peripheral blood and umbilical cord blood may be used. In addition, instead of mesenchymal stem cells, ES cells, somatic stem cells, bone cells, chondrocytes, nerve cells, or the like may be used.
Furthermore, although bone tissue has been described as an example of a living tissue, instead of this, chondrocytes other than bone, epidermal cells, dermal cells, corneal cells, gastrointestinal epithelium, endothelial cells, nerve cells, etc. Good.
[0036]
In this embodiment, BMP (bone morphogenetic protein), BDGF (bone-derived growth factor), TGF (transforming growth factor), FGF ( Cell growth factors such as fibroblast growth factor), PDGF (platelet-derived growth factor), and cytokines may be impregnated in advance.
[0037]
【The invention's effect】
As described above, according to the biological tissue replenishing material of the present invention, the method and the container for producing a biological tissue replenishing material, cells can be efficiently attached to the through holes formed in the biological tissue replenishing material, This has the effect that nutrients can be supplied to the cells during culture and waste products can be smoothly discharged from the cells. Therefore, it is necessary to produce a living tissue filling material that allows cells to grow uniformly and sound even inside the living tissue filling material and that can be rapidly and sufficiently remodeled after being filled in a defect of the living tissue. Can be.
[Brief description of the drawings]
FIG. 1 is a longitudinal sectional view showing a living tissue replacement according to an embodiment of the present invention.
FIG. 2 is a diagram illustrating a method of manufacturing a living tissue replacement using the living tissue replacement of FIG. 1;
FIG. 3 is a longitudinal sectional view showing a state in which a cell-containing liquid has been dropped into a through-hole of the living tissue filling material of FIG. 1;
FIG. 4 is a longitudinal sectional view showing a modified example of the living tissue filling material of FIG. 1;
FIG. 5 is a perspective view showing the living tissue filling material of FIG. 4;
FIG. 6 is a longitudinal sectional view showing another modified example of the living tissue filling material of FIG. 1;
FIG. 7 is a perspective view showing a container according to an embodiment of the present invention.
8 is a longitudinal sectional view showing the container of FIG.
9 is a vertical sectional view showing a protrusion formed on the bottom surface of the container of FIG.
FIG. 10 is a longitudinal sectional view showing a modification of the projection.
FIG. 11 is a longitudinal sectional view showing a modification of FIG. 8;
[Explanation of symbols]
1,12,15 Living tissue filling material 2,13 Through hole 2b Small diameter part (flow restriction part)
10, 14 Container 10a Bottom surface 11 Projection (flow restriction part)
15a Restriction part (flow restriction part)

Claims (4)

A block-shaped living tissue replacement material, comprising a through-hole,
A living tissue filling material provided with a flow restricting portion for restricting the flow of cells flowing into the through hole. The living tissue replacement material according to claim 1, wherein the flow restricting portion is configured by a small diameter portion in which the diameter of the through hole is partially reduced. A block-shaped living tissue replenishing material having a through-hole is arranged so that the through-hole extends along the vertical direction, and the lower end of the through-hole is incompletely closed with a stopper, and contains cells from above the through-hole. A method for producing a biological tissue replacement body in which a drained liquid flows down. It is a container used for the manufacturing method of the living body tissue complement according to claim 3,
On the bottom surface, provided with a projection provided at a position corresponding to the through-hole formed in the living tissue replacement material,
A container wherein the diameter of the projection is formed at an upper end smaller than the diameter of the through hole and at a lower end larger than the diameter of the through hole.

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