JP4347554B2 - Specimen manufacturing method for infection test using biological tissue filling material - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention provides, for example, a specimen manufacturing method for an infection test using a biological tissue filling material, which is optimal for performing an infection test on a biological tissue complement used when regenerating a biological tissue defect such as a bone defect. It is about.
[0002]
[Prior art]
In recent years, by replacing a biological tissue defect material such as a bone defect caused by bone tumor extraction or trauma etc. with a bone tissue replacement material or the like, the bone tissue or the like is regenerated to regenerate the bone tissue or the like. It has become possible to repair.
[0003]
For example, hydroxyapatite (HAP) and tricalcium phosphate (TCP) are known as bone prosthetic materials, but they are made of a calcium phosphate porous material such as β-TCP from the viewpoint of leaving no foreign matter in the body. Scaffolding material is used. When this β-TCP is brought into contact with the bone cells of the bone defect portion, so-called remodeling is performed by osteoclasts and osteoblasts, and the bone filling material compensated for the bone defect portion is self-generated over time. It is replaced with bone (for example, see Non-Patent Document 1).
[0004]
On the other hand, in order to increase the repair speed of the bone defect part after surgery, the bone grafting material is not used as it is, but bone marrow mesenchymal cells collected from the patient are cultured together with the bone grafting material, and then the bone defecting part is used. Compensation is being done. In this case, the bone substitute is cultivated in the cultured bone filling material. Prior to filling, for example, whether there is no infection such as HIV (human AIDS virus), HBV (hepatitis B virus), etc. Therefore, it is desired to supplement the bone filling material after confirming that there was no virus infection.
[0005]
[Non-Patent Document 1]
Uemura et al., “Tissue engineering in bone using biodegradable β-TCP porous material -Osferion, a new material that increases strength in vivo”, Medical Asahi, Asahi Shimbun, October 1, 2001, No. 30 Volume 10, No. 10, p. 46-49
[0006]
[Problems to be solved by the invention]
However, when such an infection test is performed, it is possible to take out the floating microorganisms from the broth culture medium to be supplemented, and to perform the test, but in this way, it adheres to the bone complement to be examined. Since the adherent cells themselves are different from the adherent cells attached to the bone substitute to be supplemented, it is necessary to inspect a large number of culture solutions, which increases the cost.
[0007]
In addition, it is conceivable to collect cells from the surface of the bone prosthesis before supplementation, but there is a problem that it is necessary to take infection prevention measures at the time of collection, and the number of work steps increases.
Accordingly, the present invention provides a specimen manufacturing method for infectious examination using a biological tissue filling material that makes it possible to accurately examine the presence or absence of infection of microorganisms such as bacteria and viruses in the biological tissue complement to be supplemented. It is.
[0008]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, a method for producing a specimen for infection test using the biological tissue filling material of the present invention is a porous bioaffinity for producing a biological tissue filling material by attaching cells by attaching a cell-containing liquid. Another biological tissue filling material (for example, the bone grafting material 11 in the embodiment) is placed on the surface of the biological tissue filling material (for example, the bone grafting material 10 in the embodiment) made of a material, and cells are attached to these. Manufacturing a plurality of biological tissue replacements (for example, bone replacements 10A and 11B in the embodiment), separating at least two biological tissue replacements, and using the separated one as a specimen for an infection test. Features.
By configuring in this way, when using another biological tissue complement as a specimen, it is sufficient to separate them.
[0014]
According to a second aspect of the present invention, in the specimen manufacturing method for an infection test using the biological tissue filling material, one of the biological tissue filling materials is a granular biological tissue filling material (for example, the bone filling material in the embodiment). 11).
By comprising in this way, the biological tissue complement which is discarded after a test | inspection becomes a granular small thing.
[0015]
The invention described in claim 3 is characterized in that, in the specimen manufacturing method for an infection test using the biological tissue filling material, both of the biological tissue filling materials are block-shaped biological tissue filling materials.
By comprising in this way, it becomes possible to use the biological tissue filling material body in which the joint surface of each biological tissue filling material has the same shape.
[0016]
According to a fourth aspect of the present invention, there is provided a porous bioaffinity for producing a biological tissue complement by attaching cells by attaching a cell-containing liquid in the specimen manufacturing method for an infection test using the biological tissue filling material. At least two living tissue filling materials made of materials (for example, bone filling materials 12 and 13 in the embodiment) are adjacent to each other, and one surface of the two adjacent living tissue filling materials (for example, one surface F in the embodiment) is polymerized. A plurality of biological tissue filling bodies (for example, the bone filling bodies 12A and 13B in the embodiment) by attaching cells to the plurality of biological tissue filling materials in the clamped state to attach the cell-containing liquid. Is manufactured, and one biological tissue filling body (for example, bone filling body 13B in the embodiment) separated by releasing the clamp is used as a specimen for infection test. And wherein the Rukoto.
By constructing in this way, when producing a biological tissue complement, both can be produced under the same conditions as when they are integrated, so that the identity of both cultured cells when separated is maximized. Can be increased.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below with reference to the drawings.
First, the process of manufacturing a bone graft using a bone graft material will be schematically described. As shown in FIG. 3, bone marrow fluid is collected from a patient's iliac or the like, and the collected bone marrow fluid is centrifuged to extract bone marrow cells having a high specific gravity.
[0018]
The extracted bone marrow cells are put into a culture container together with a previously prepared medium and mixed. Part of the medium is removed and sent for infection testing.
After this, the mixed bone marrow fluid and medium are maintained at the culture conditions such as a predetermined temperature (for example, 37 ± 0.5 ° C.) and CO ₂ concentration (for example, 5%), and are maintained under a constant culture condition for a predetermined time. Cells are primary cultured. The medium is discarded from the culture container at a predetermined replacement time during the cell culture. Then, the culture medium is mixed again and the culture process is repeated. Part of the discarded medium is sent for infection testing.
[0019]
When the predetermined culture period ends, the medium is discarded from the culture container, and then a proteolytic enzyme such as trypsin is charged and mixed in the culture container. As a result, the mesenchymal stem cells that have grown on the bottom surface of the culture vessel are detached from the bottom surface of the main culture vessel, and the exfoliated mesenchymal stem cells are extracted by being subjected to a centrifuge. .
[0020]
The extracted mesenchymal stem cells are adjusted in the number of cells and then mixed in a culture container in which a bone filling material and an appropriate medium are put. In practice, mesenchymal stem cells are attached to the bone grafting material and put into the medium. Then, in the same manner as described above, by maintaining the mixed mesenchymal stem cells and the medium at a predetermined temperature (for example, 37 ± 0.5 ° C.) and a culture condition such as CO ₂ concentration (for example, 5%), Cells are subcultured under constant culture conditions for a predetermined time.
[0021]
In the secondary culturing step, similarly to the primary culturing step, the medium is periodically exchanged, and a part of the medium to be input and a part of the medium to be discarded are sent to the infection test. Then, when a predetermined culture period has passed, sample extraction for shipping quality inspection and infection inspection is performed, and the manufactured bone filling material is sealed and provided as a product.
[0022]
Here, as the medium, for example, a mixture of MEM (Minimal Essential Medium), FBS (Fetal Bovine Serum), and antibiotics at a predetermined blending ratio is used. Further, growth factors such as cytokines, concentrated platelets, BMP, FGF, TGF-β, IGF, PDGF, VEGF, HGF, and a substance that contributes to growth, such as a combination thereof, may be mixed. Further, hormone agents such as estrogen and nutrients such as vitamins may be mixed. Human serum may be used instead of fetal bovine serum.
Furthermore, as the antibiotic, any antibiotic such as cephem, macrolide, tetracycline, fosfomycin, aminoglycoside, and new quinolone can be employed in addition to the penicillin antibiotic.
[0023]
Next, a biological tissue filling material according to a reference example of the present invention will be described.
FIG. 1 is a perspective view showing a bone grafting material of a first reference example of the present invention. This bone filling material (living tissue filling material) 1 is manufactured by the method (mechanochemical method) disclosed in Japanese Patent No. 2597355. Usually, the bone grafting material 1 is formed in a rectangular parallelepiped shape, a pellet shape, and a granule shape, and is used for filling a bone defect portion after bone marrow fluid is applied. In this embodiment, an external force is applied. It can be easily divided into at least two. Here, the main component of the bone grafting material 1 is β-TCP (beta-tricalcium phosphate which is a porous biocompatible material).
The bone prosthetic material 1 may be any material as long as it is a material having affinity for living tissue, and more preferably a bioabsorbable material. In particular, porous ceramics having biocompatibility, collagen, polylactic acid, polyglycolic acid, hyaluronic acid, or a combination thereof may be used. Further, a metal such as titanium may be used.
[0024]
As shown in FIG. 1, the bone grafting material 1 is formed in a substantially rectangular parallelepiped shape having a long one side, and a cut portion 2 that facilitates division is provided at a divided portion thereof, that is, a central portion in the longitudinal direction.
The cut portion 2 is formed over substantially the entire circumference, leaving a connecting portion 3 (shown by hatching) having a triangular cross section at the center in the longitudinal direction of the cuboid bone filling material 1. As described above, since the connecting portion 3 having a small area is left in the corner portion of the square cross-sectional shape, it can be easily folded without trouble when an external force is applied. Therefore, it is possible to secure a large surface area of the portions facing each other with the notch 2 interposed therebetween, thereby ensuring a large adhesion area of mesenchymal stem cells.
[0025]
Here, the shape of the notch 2 is not limited to that described above as long as the bone grafting material 1 can be divided into two by an external force. For example, like the incision 2 shown in FIG. 2, the rectangular bone-shaped bone grafting material 1 is formed over the entire circumference leaving the connecting portion 4 (shown by hatching) having a rectangular cross section at the center in the longitudinal direction. May be.
As shown in FIG. 2, when the connecting portion 4 having a small cross-sectional square shape is left in the central portion of the square cross-sectional shape, the bone prosthetic material 1 becomes symmetrical around the connecting portion 4 and is folded in two. There is no need to select the direction, and the mounting posture during culture is not directional and the operation is easy to perform.
[0026]
Therefore, according to the first reference example , the bone grafting material 1 is used in the secondary culturing step, and after a predetermined culture period has elapsed, an external force is applied so that the bone grafting material 1 is connected to each cut portion 2. Can be divided by the connecting portion 3 or the connecting portion 4. Thereby, the two bone filling bodies 1A and 1B cultured under the same conditions can be obtained.
As a result, as shown in FIG. 4, it is possible to use one as a bone prosthesis 1A for filling the defect portion 5 and the other as a bone prosthesis 1B as a specimen for infection test. Therefore, the conditions of the cells of the bone substitute 1A for supplementation and the cells of the bone substitute 1B for specimen can be made to completely match, and the examination result of one of the bone substitutes 1A can be used as the supplementary result as it is. The result is reliable and accurate.
[0027]
In addition, since the bone replacement body 1A for replacement does not need to be inspected before being replaced, the cleanliness is not impaired, and the reliability of the bone replacement body 1A can be improved. The bone substitute 1B, which is one specimen for which the infection test has been completed, is stored as it is, so that it becomes a sample for knowing the degree of culture of the supplemented bone substitute 1A in the living body and has a high useful value. .
[0028]
FIG. 5 is a perspective view showing a bone grafting material of a second reference example of the present invention.
In this reference example , the bone grafting material 1 is composed of a bone grafting material body 6 and an inspection piece 7 protruding from the bone grafting material body 6, and can be divided into at least two parts when an external force is applied. It is what.
More specifically, an inspection piece 7 having a trapezoidal cross section is provided on one side edge of the rectangular bone-shaped bone prosthesis main body 6, and the cross-sectional area of the inspection piece 7 decreases toward the base. It is formed as a constricted portion 8. Therefore, the inspection piece 7 can be easily folded at the constricted portion 8.
[0029]
Therefore, according to this reference example , in addition to the effects of the above reference example , the bone substitute 1 after the secondary culture is completed can be easily replaced by folding the test piece 7 and the bone substitute main body 6A for supplementation and the specimen. It becomes possible to divide and obtain the inspection piece 7B used for the purpose. In addition, since the inspection piece 7B is smaller than the bone substitute main body 6A, waste of the bone filling material 1 to be discarded after the inspection can be reduced as much as possible.
[0030]
Note that the cross-sectional shape of the test piece 7 is not limited to a trapezoidal cross-section, and as shown in FIG. Concave portions 9 and 9 are formed on both sides of the connecting portion, and a constricted portion 8 is provided here so that the constricted portion 8 can be divided into a bone prosthesis body 6′A and an inspection piece 7′B. May be.
[0031]
In addition, according to the method for producing the bone substitute for specimen and supplement using the bone substitute material 1 having the special shape described above, the bone substitute 1A or bone substitute to which the cultured mesenchymal stem cells are attached. The body body 6A, 6′A is shipped and the other divided bone prosthesis 1B used for the specimen or the examination pieces 7B, 7′B are inspected for infection of bacteria, etc. at the same time, and based on the examination result of the specimen In addition, since the bone replacement body 1A or the bone replacement body main bodies 6A and 6′A can be quickly replaced with the arrival of the bone replacement body 1A, a series of operations from the cultivation of the bone replacement body to the replacement can be quickly performed.
[0032]
Next, a specimen manufacturing method for infection testing using a bone grafting material will be described with reference to FIGS.
In the specimen manufacturing method for the infection test using the bone filling material of the reference example described above, a step of applying an external force is necessary to divide the bone filling material after the secondary culture. In this manufacturing method, a specimen for infection test can be manufactured more easily.
[0033]
As shown in FIG. 7, a granular bone grafting material 11 is placed on the upper surface of a cubic bone grafting material 10, and mesenchymal stem cells are attached to these by secondary culture to form a cubic bone grafting material. The body 10A and the granular bone filling body 11B are manufactured, the granular bone filling body 11B is separated, and the separated granular bone filling body 11B is used as a specimen for the infection test. Therefore, since the secondary culture is completed, the cube-shaped bone filling body 10B is used for filling, and the granular bone filling body 11B can be taken out as it is to obtain a specimen for an infection test. This eliminates the need for this work and facilitates the separation work in a sterile bag. Further, by using the granular bone filling body 11B as a specimen, the bone filling body 11B to be discarded after the examination becomes small, so that waste can be minimized. Further, the granular bone grafting material 11B is advantageous in that the ground contact portion is almost a point, so that no unattached portion of the mesenchymal stem cells remains at the taken-out placement location.
[0034]
Further, as shown in FIG. 8, a plurality of granular bone grafting materials 11, 11... Serving as specimens for infection testing may be arranged on a plurality of surfaces of the bone grafting material 10 for filling. Good. In this way, it is possible to easily obtain a plurality of granular bone filling bodies 11B, 11B,... That will serve as specimens for infection testing, and confirm the culture status of the bone filling body 10A after filling. As a sample to be obtained, there is a merit that a plurality of granular bone filling bodies 11B can be obtained for each surface of the cubic bone filling body 10A, for example.
Here, instead of the granular bone filling material 11, a block-like bone filling material can be placed and used in a stacked manner. If it does in this way, there exists a merit which can use the bone grafting material 10 of the same shape, for example.
[0035]
Next, another embodiment of a specimen manufacturing method for an infection test using a bone grafting material will be described with reference to FIGS.
As shown in FIGS. 9 and 10, the bone grafting materials used in the specimen manufacturing method for this infection test are both bone-shaped bone grafting materials 12, 13 as shown in FIGS. 9 and 10. Each of the bone grafting materials 12 and 13 has a shape capable of aligning one surface F with the other bone grafting material. In this embodiment, each bone grafting material is a rectangular parallelepiped having the same cross-sectional shape. Is larger than the bone grafting material 13. Both may be the same shape of bone substitute material.
[0036]
First, the two bone grafting materials 12 and 13 are made adjacent to each other, and the surfaces F of the two adjacent bone grafting materials 12 and 13 are superposed to be pressed by a clamping device (not shown) to clamp them. Then, in the secondary culture step, in the secondary culture step, seeding is performed using both the clamped bone substitutes 12 and 13, and when the predetermined culture period has elapsed, the clamp is released by the clamp device. One bone filling body 13B is used as a specimen for infection test. The other bone filling body 12A is used for filling. As a result, when producing both bone substitutes 12A and 13A, both can be manufactured under the same conditions as when they are integrated, so that the identity of both cultured cells when separated is maximized. Therefore, the identity of the bone substitute 12A to be compensated and the bone substitute 13B for the specimen is ensured, and the reliability can be improved.
[0037]
The present invention is not limited to the above embodiment. For example, the bone grafting material 1 shown in FIGS. 1 and 2 has a structure that can be divided into two parts, but has a structure that can be divided into three or more parts. It may be.
In the above embodiment, bone is used as an example of living tissue, and the case of culturing mesenchymal stem cells extracted and cultured from bone marrow fluid has been described, but extracted from peripheral blood and umbilical cord blood as well as bone marrow fluid. You may decide to do it.
[0038]
【The invention's effect】
As described above, according to the specimen manufacturing method for infection testing using the biological tissue filling material of the present invention, when other biological tissue filling material is used as a specimen, it is only necessary to separate them. Therefore, there is an effect that the work is easy to perform.
[0045]
According to the second aspect of the present invention, the biological tissue filling material to be discarded after the examination becomes a small granular material, so that it is possible to minimize waste.
[0046]
According to the invention described in claim 3, since it is possible to use a living tissue filling material having the same shape as the mating surface , both are subjected to the same culture condition as compared with the case of using a different shape. There is an effect that can be put. In addition, when the body tissue complement to be supplemented and the specimen have the same shape, the state of the compensated one can be most accurately reproduced when knowing the post-operative progress from the specimen, and the value of the sample is great. effective.
[0047]
According to the invention described in claim 4, when producing a biological tissue complement, since both can be produced under the same conditions as in the case where they are integrated, the identity of both cultured cells when separated is obtained. Therefore, it is possible to enhance the reliability by ensuring the identity between the body tissue complement to be supplemented and the specimen.
[Brief description of the drawings]
FIG. 1 is a perspective view showing a bone grafting material of a first reference example of the present invention.
FIG. 2 is a perspective view of a bone grafting material showing another embodiment of FIG.
FIG. 3 is an explanatory diagram of a culture process according to the embodiment of the present invention.
FIG. 4 is an explanatory diagram showing a situation in which the bone substitute after culturing according to the first reference example of the present invention is used for filling and for a specimen.
FIG. 5 is a perspective view showing a bone grafting material of a second reference example of the present invention.
6 is a side view showing another embodiment of FIG. 5. FIG.
FIG. 7 is a perspective view of a bone filling material used in a specimen manufacturing method for infection testing using the biological tissue filling material according to the present invention.
8 is a perspective view showing another embodiment of FIG.
FIG. 9 is an explanatory view showing a bone filling material used in a specimen manufacturing method for infection testing using a biological tissue filling material according to the present invention.
FIG. 10 is an explanatory view showing a specimen manufacturing method for an infection test using another biological tissue filling material according to the present invention.
[Explanation of symbols]
1,10,11,12,13 Bone prosthetic material (living tissue prosthetic material)
2 Incision part 6, 6 'Bone prosthesis body 7, 7' Inspection piece 8 Constriction part 10A, 11B Bone prosthesis body (biological tissue supplement)
12A, 13B Bone replacement (biological tissue replacement)

Claims (4)

Other biological tissue filling materials are placed on the surface of a biological tissue filling material made of a porous biocompatible material for producing a biological tissue filling material by attaching cells by attaching a cell-containing liquid, and cells are placed on these materials. Infection using a biological tissue filling material, wherein a plurality of biological tissue filling materials are produced by attachment, and at least two biological tissue filling materials are separated, and the separated one is used as a specimen for infection testing Specimen manufacturing method for examination. The specimen manufacturing method for an infection test using the biological tissue filling material according to claim 1 , wherein one of the biological tissue filling materials is a granular biological tissue filling material. 2. The specimen manufacturing method for an infection test using the biological tissue filling material according to claim 1 , wherein both of the biological tissue filling materials are block-shaped biological tissue filling materials. Adhering at least two biological tissue filling materials made of a porous biocompatible material for producing a biological tissue filling material by attaching cells by attaching a cell-containing liquid, and adjoining one surface of these two adjacent biological tissue filling materials Clamping in a polymerized state, attaching a cell-containing liquid to multiple clamped tissue replacement materials to produce cells by attaching cells, and separating by releasing the clamp A specimen manufacturing method for an infection test using a biological tissue filling material, characterized in that the single biological tissue complement is used as a specimen for an infection test.

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