JP2003320007A - Method of manufacturing living tissue filling body - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To attach a sufficient quantity of mesenchymal stem cells to a living tissue filling material in a limited period of time during an operation. <P>SOLUTION: The method is for manufacturing the living tissue filling body to be filled in a deficit part of a living tissue of a patient. The method comprises a step S1 to collect humor from the patient, a step S2 to extract at least a concentrate body including mesenchymal stem cells from the collected humor, a step S3 to dilute the extracted concentrate body with a diluent, and a step S4 to spray the diluted concentrate body on the living tissue filling material. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a body tissue filling body.

[0002]

2. Description of the Related Art In recent years, a living tissue such as a bone is regenerated by filling a living tissue defective portion such as a bone defective portion caused by excision of a bone tumor or trauma with a living tissue filling material such as a bone filling material. It has become possible to repair a defect in a biological tissue. Hydroxyapatite (H
AP) and tricalcium phosphate (TCP) are known, but from the idea that no foreign matter remains in the body, for example,
A scaffold made of calcium phosphate porous material such as β-TCP is used. When β-TCP is brought into contact with bone cells in a bone defect, osteoclasts eat β-TCP and osteoblasts form new bone, so-called remodeling is performed. That is, the bone filling material filled in the bone defect portion is replaced with autogenous bone over time.

By the way, in order to increase the speed of repair of a bone defect portion after surgery, the bone filling material is not used as it is, but bone marrow fluid collected from a patient is applied to the bone filling material to fill the bone defect portion. Is being done.

[0004]

In this case, bone marrow fluid containing a large amount of mesenchymal stem cells is collected from a patient, and a bone filling material is dipped in this bone marrow fluid to fill the bone defect portion. However, there is a problem that a sufficient amount of mesenchymal stem cells cannot be attached to the bone filling material by merely immersing the bone filling material in the bone marrow fluid. Further, in order to attach a sufficient amount of mesenchymal stem cells to the bone prosthetic material, it has been considered that the proliferated mesenchymal stem cells are adhered to the bone prosthetic material by culturing the extracted bone marrow fluid. . However, since the culture of mesenchymal stem cells requires a period of about 2 weeks, patients should have at least two visits, one for collection of bone marrow fluid before surgery and one for surgery. There was the inconvenience that it was required, and the time and physical burdens were heavy.

The present invention has been made in view of the above-mentioned circumstances, and a living tissue capable of adhering a sufficient amount of mesenchymal stem cells to a living tissue repair material within a limited time during surgery. It is intended to provide a method for manufacturing a filling body.

[0006]

In order to achieve the above object, the present invention provides the following means. The invention according to claim 1 is a method for producing a body tissue filling body for filling a body tissue defect portion of a patient during surgery, comprising a step of collecting a body fluid from the patient, and at least mesenchymal stem cells from the collected body fluid. Provided is a method for producing a biological tissue filling body, the method comprising: a step of extracting a concentrated body containing the biological tissue; a step of diluting the extracted concentrated body with a diluent; and a step of applying the diluted concentrated body to a biological tissue filling material. To do. According to the present invention, the collected body fluid is not directly attached to the biological tissue filling material, but the concentrated body containing mesenchymal stem cells is extracted from the body fluid, so that the biological tissue filling containing many components contributing to growth is added. The body can be manufactured. At this time, by performing the step of diluting the concentrated body with a diluent,
A highly viscous condensate can be made into a low viscous liquid,
By merely spraying this on the biological tissue filling material, it becomes possible to uniformly attach the mesenchymal stem cells to the biological tissue filling material and to permeate the inside. Here, the body fluid is bone marrow fluid, peripheral blood, cord blood, or the like. The concentrate is obtained by removing at least one unnecessary component of blood cells or tissue fluid from the collected body fluid, and is, for example, a liquid or gel-like body containing at least mesenchymal stem cells and the like.

According to a second aspect of the present invention, in the manufacturing method according to the first aspect, before the diluted concentrate is sprayed on the living tissue filling material, the living tissue filling material is formed into a shape of the living tissue defective portion. There is provided a method for producing a biological tissue filling body which is processed according to the above. According to the present invention, the biological tissue filling material before the concentrate is sprayed is processed to match the shape of the biological tissue defective portion, so that the biological tissue is not wasted or damaged. It becomes possible to manufacture a biological tissue filling body that exactly fits the defect.

The present invention according to claim 3 is a method for producing a body tissue filling body for filling a body tissue defect portion of a patient during an operation, comprising a step of collecting body fluid from the patient and at least an interval from the body fluid collected. Provided is a method for producing a biological tissue filling body, which comprises a step of extracting a concentrated body containing leaf stem cells and a step of attaching the extracted concentrated body to a living tissue filling body. According to this invention, since the concentrate containing mesenchymal stem cells extracted from the collected body fluid is attached to the biological tissue supplement, it is possible to produce a biological tissue supplement having a large amount of components contributing to growth. Become.

According to a fourth aspect of the present invention, in the manufacturing method according to the third aspect, before attaching the concentrated body to the living tissue filling material, the living tissue filling material is adjusted to the shape of the living tissue defective portion. Provided is a method for producing a living tissue filling body to be processed. According to this invention, the living tissue filling material before the attachment of the concentrate is processed to match the shape of the living tissue defective portion, so that the living tissue defective without waste of cells or damage to the cells can be obtained. It is possible to manufacture a biological tissue filling body that fits exactly to the part.

[0010] The invention according to claim 5 is for a predetermined period until the living tissue filling body manufactured by the manufacturing method according to any one of claims 1 to 4 is transplanted into a living tissue defective portion of a patient. Provided is a method for producing a body tissue filling body which keeps temperature and humidity at the same temperature and humidity. According to the present invention, the biological tissue filling body manufactured by the manufacturing method according to any one of claims 1 to 4 is kept warm and moist, so that it can be maintained in a state protected from temperature change and drying.
Therefore, even if the operation period is extended, it is possible to provide the body tissue filling body in a state suitable for filling the body tissue defect portion.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION A method for manufacturing a body tissue filling body according to an embodiment of the present invention will be described below with reference to FIGS. 1 and 2. The manufacturing method according to the present embodiment is, for example, a method for manufacturing a bone filling body for filling a bone defect portion formed in bone as a kind of biological tissue.

The method for manufacturing a bone prosthesis according to this embodiment is
As shown in FIG. 1, it includes a bone marrow fluid collecting step S1, a condensate extracting step S2, a diluting step S3, and a spraying step S4. Concentrated body extraction step S2
Includes a separation step S5 and an unnecessary component removal step S6.

As shown in FIG. 2, the bone marrow fluid collecting step S1 is a step of collecting the bone marrow fluid 2, which is a body fluid, from the patient's body A, for example, the iliac bone, by the syringe 1. The separation step S5 is performed by, for example, a centrifugation method, and the bone marrow fluid 2 collected in the above bone marrow fluid collection step S1 is used.
As shown in FIG. 3, (a) is placed in a test tube-shaped centrifuge container 3 and (b) is rotated by a centrifuge 4, so that (c) the centrifugal container 3 is placed outward in the radial direction of rotation of the centrifuge container 3. In this step, the concentrated body 5 having a large specific gravity can be separated on the bottom side, and the unnecessary component 6 such as the tissue fluid having a smaller specific gravity can be separated on the upper side. The unnecessary component removing step S6
After separating the condensate 5 from the bone marrow fluid 2 in this way,
As shown in FIG. 4, the step is to take out only the concentrated body 5 by discarding the unnecessary component 6 in the supernatant.

In order to remove blood cells from the bone marrow fluid 2, an antibody complex is added to the bone marrow fluid 2 so that red blood cells in the bone marrow fluid 2 are bound to unnecessary cells through the antibody complex to form a rosette. It is conceivable to add a medium for specific gravity separation and centrifuge. In this case, unnecessary cells that have become rosettes may be precipitated and the concentrate 5 containing the desired mesenchymal stem cells may be recovered from the upper layer of the medium for specific gravity separation.

The diluting step S3 is, as shown in FIG.
This is a step of reducing the viscosity of the concentrated body 5 by adding an arbitrary diluent 7 such as physiological saline to the concentrated body 5 extracted and recovered as described above. In the spraying step S4, as shown in FIG. 6, the diluted solution of the concentrated body 5 obtained as described above is uniformly sprayed on the surface of the bone prosthetic material 9 by any spraying means 8 such as a spray. It is a step to do. As the bone filling material 9, for example,
A porous body made of β-TCP manufactured by the method disclosed in Japanese Patent No. 2597355 is used.

As described above, according to the method for manufacturing a bone filling material according to the present embodiment, the concentrate 5 containing mesenchymal stem cells etc. from which the unnecessary component 6 not involved in bone formation is removed is sprayed on the bone filling material 9. The bone filling body capable of promptly repairing the bone defect portion can be manufactured by the simple method. That is,
Since each step is completed in a short time, collection of bone marrow fluid 2 to production of a bone filling material can be performed in a short time. In addition, the bone substitute manufactured in this way is
Since the unnecessary component 6 is not added, a large amount of components contributing to bone formation can be added, and the bone defect portion can be repaired at an early stage.

Next, a method for manufacturing a biological tissue filling body according to a second embodiment of the present invention will be described below with reference to FIG. In the description of this embodiment, the above-mentioned first
The same reference numerals are given to portions having the same configuration as the manufacturing method according to the embodiment, and the description will be simplified. Similar to the manufacturing method according to the first embodiment, the manufacturing method according to the present embodiment is also a method for manufacturing a bone filling body for filling a bone defect portion, and in addition to the first manufacturing method, a concentrated body Before the spraying step S4 of 5, there is provided a determination step S7 regarding whether or not to process the bone filling material 9, and a processing step S8.

In the judgment step S7, it is judged whether or not the bone filling material 9 needs to be processed, and if it is necessary, the processing proceeds to the processing step S8, and if it is not necessary, the processing proceeds to the spraying step S4. Has become. The bone filling material 9 needs to be processed, for example, when a porous block-shaped bone filling material 9 made of β-TCP is provided, a bone filling body made of the bone filling material 9 is required. Since the volume of the bone defect portion to be actually filled is smaller than that of the bone filling material 9, the bone filling material 9
In the case where the bone filling material 9 does not enter the bone defect portion, or the shape of the bone filling material 9 does not match the shape of the bone defect portion and the bone filling material 9 protrudes from the bone surface. Further, it is judged that the bone filling material 9 does not need to be processed when the bone filling material 9 is in a granular shape,
This is the case when it is in the form of a sufficiently small porous block.

In the processing step S8, the bone filling material 9 is subjected to processing such as cutting and boring. In addition, the powder of the bone filling material 9 generated as a cutting dust is also recovered if necessary,
It is sent to the spraying step S4 together with the processed bone filling material 9.

Here, in the example shown in FIG. 7, the judgment step S7 and the processing step S8 are arranged between the dilution step S3 and the spraying step S4, but the present invention is not limited to this, and these steps are not limited thereto. The determination step S7 and the processing step S8 may be arranged at any position before the spraying step S4.

According to the method for manufacturing a bone prosthesis of this embodiment having the above-described structure, the same operational effect as that of the manufacturing method of the first embodiment can be obtained, and the method can be fitted to a bone defect portion. It is possible to manufacture a bone substitute body that does this. Therefore, the bone filling body can be stably held in the bone defect portion, and stable bone formation can be achieved. Further, the concentrate 5 is added to the bone filling material 9 powder generated as shavings.
It is also possible to embed the bone filling material made of these powders in the gap generated when the bone filling material is filled in the bone defect portion.

Further, since the processing step S8 for processing the bone filling material 9 is provided before the spraying step S4 of the concentrated body 5, even if the bone filling material 9 is cut or punched, cell damage is not caused. Therefore, it is possible to rapidly manufacture a sound bone substitute that fits the bone defect portion.

Next, a method for manufacturing a body tissue filling body according to a third embodiment of the present invention will be described below with reference to FIG. The manufacturing method according to the present embodiment is also a method for manufacturing a bone prosthesis, and the dilution step S3 of the manufacturing methods according to the first and second embodiments described above is eliminated, and an application step S9 is provided instead of the spraying step S4. It is a thing.
It should be noted that the manufacturing step according to the second embodiment is similar to the manufacturing step according to the second embodiment in that the processing step S8 of the bone prosthetic material 9 is present before the applying step S9, but the determination step S7 and the processing step S8 are deleted. Good. In order to adjust the viscosity of the concentrate 5, an appropriate amount of physiological saline or the like may be added to produce a paste-like concentrate 5 having a low viscosity and the paste 5 may be applied to the bone filling material 9. .

According to the method for manufacturing a bone prosthesis according to the present embodiment having the above-mentioned structure, as in the manufacturing methods according to the first and second embodiments, a sound bone prosthesis suitable for a bone defect portion can be obtained. The body can be manufactured quickly.

The manufacturing method according to these embodiments is shown in FIG.
Can be performed during surgery, as shown in. That is, when a patient is laid on a bed and an operation is started, a treatment step for opening a diseased part such as a lesion (process P).
Prior to the start of 1), a bone marrow fluid collecting step S1 included in the method for manufacturing a biological tissue filler according to the first to third embodiments is performed (process P2). Then, these processes P1 and P2 are simultaneously performed in parallel,
In the process P1, a bone defect portion is exposed by scraping a lesion such as a bone tumor portion.

On the other hand, in the process P2, the dilution step S
3 or impurity removal step S6 is in progress. Then, based on the volume and the shape of the bone defect portion which is clarified in the process P1, the process P1 requests the process P2 to process, and the bone filling material 9 is processed.
In practice, a doctor who scrapes a bone defect portion cuts the bone prosthetic material 9 in accordance with the bone defect portion, so that the bone prosthetic material 9 suitable for the bone defect portion is formed. Thereafter, the bone filling body manufactured through the spraying / application steps S4 and S9 is returned to the process P1. Then, the bone prosthesis thus constructed is transplanted to the bone defect portion, and the epithelial tissue is sutured to complete the operation.

As described above, according to the manufacturing method of each of the above-described embodiments of the present invention, the bone filling body can be manufactured in a short time after the bone marrow fluid 2 is collected, so that the bone filling body can be prepared during the transplant operation. Can be manufactured and transplanted. That is, the patient does not need to collect the bone marrow fluid 2 prior to the transplant operation, and can be completed in one visit. Further, since the bone prosthesis can be manufactured during surgery, a bone prosthesis that fits exactly can be manufactured based on the accurate shape and size of the bone defect portion. That is, the shape of the bone defect part can be judged to some extent by CT diagnosis or MRI diagnosis before surgery, but at the stage when the affected part is actually opened and the tumor or the like is scraped,
Sometimes it becomes clear. In this case, the doctor can process the bone filling body while looking at the bone defect portion. Moreover,
Since the bone filling material 9 before the concentrated body 5 is processed is processed according to the shape of the bone defect portion and the like, the manufactured bone filling material is not excised, so that healthy bones without cell damage or the like are processed. A filler can be obtained.

Depending on the operation, there are cases in which the required time becomes indefinite such that the operation process is interrupted or the time is extended. That is, the treatment process P1
There is a case where the bone filling body manufacturing process P2 that is performed in parallel with the above is completed first and the bone filling body is manufactured.
In this case, as shown in FIG. 10, it is judged whether or not the preparation for transplantation is completed (step S10), and if the preparation is completed, the procedure is returned to the treatment process P1 and the preparation is not completed. For example, the produced bone substitute is placed under an environment of suitable temperature and humidity, for example, temperature 37 ° C., humidity 70-9.
It may be stored in a 5% heat retaining / humidifying box (heat retaining / humidifying step S11). As a result, it is possible to prevent damage of the mesenchymal stem cells contained in the bone filling material due to drying or the like.

In the present embodiment, the description has been made with respect to bone as a living tissue, but instead of this, chondrocytes other than bone, epidermal cells, dermal cells, corneal cells, gastrointestinal epithelium and endothelial cells, nerves, nerves, etc. The cells may be targets. Also,
Bone prosthetic material 9 made of β-TCP as the prosthetic material for biological tissue
However, instead of this, bioabsorbable materials such as HAP, TCP or collagen, PLA (polylactic acid), PGA (polyglycolic acid), PLGA (polylactic acid-polyglycolic acid copolymer) are adopted. Good.

Further, the bone marrow fluid 2 containing a large amount of mesenchymal stem cells was collected and a living tissue supplement was produced from the bone marrow fluid 2. Instead of this, mesenchymal cells separated from peripheral blood and cord blood were used. You may decide to manufacture a biological tissue filling body using a stem cell. In addition, cytokine, platelet concentrate, BMP, F
GF, TGF-β, IGF, PDGF, VEGF, HG
The biological tissue filler may be manufactured by mixing F, a substance that contributes to growth such as a composite thereof, or the like.
Further, a substance that contributes to the above growth may be used as the diluent.

[0031]

As described above, according to the method for producing a biological tissue filling body according to the present invention, the living tissue filling body can be produced during the operation, so that the patient's time and physical burden can be reduced. Can be reduced. In addition, since a living tissue filling body is manufactured by a component that contributes to the formation of a target tissue, for example, a concentrate containing a large amount of mesenchymal stem cells, a living tissue filling body capable of promptly repairing a defect in living tissue is manufactured. There is an effect that can be.

[Brief description of drawings]

FIG. 1 is a flowchart illustrating a method for manufacturing a biological tissue filling body according to a first embodiment of the present invention.

FIG. 2 is a schematic diagram illustrating a sampling step in the manufacturing method of FIG.

FIG. 3 is a schematic diagram illustrating a separation step in the manufacturing method of FIG.

FIG. 4 is a schematic diagram illustrating an unnecessary component removing step in the manufacturing method of FIG.

5 is a schematic diagram illustrating a dilution step in the manufacturing method of FIG.

FIG. 6 is a schematic diagram illustrating a spraying step in the manufacturing method of FIG.

FIG. 7 is a flowchart illustrating a method for manufacturing a biological tissue filling body according to a second embodiment of the present invention.

FIG. 8 is a flowchart illustrating a method for manufacturing a biological tissue filler according to a third embodiment of the present invention.

FIG. 9 is a flowchart illustrating a surgical procedure to which the manufacturing method of the present invention is applied.

FIG. 10 is a flowchart showing a case where the method for manufacturing a biological tissue filling body according to another embodiment of the present invention is applied to surgery.

[Explanation of symbols]

2 Bone marrow fluid (body fluid) 5 Concentrate 7 Diluent 9 Bone filling material (living tissue filling material) S1 sampling step S2 extraction step S3 dilution step S4 spraying step S8 processing step S11 heat retention / moisturization step

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Ryoji Saito             2-43 Hatagaya, Shibuya-ku, Tokyo Ori             Inside Npus Optical Industry Co., Ltd. F-term (reference) 4C081 AA14 AB04 CD34 CF011                       DA01 DC03 EA02

Claims (5)

[Claims] 1. A method for producing, during surgery, a body tissue filling body for filling a body tissue defect portion of a patient, the method comprising a step of collecting a body fluid from the patient, and a concentration containing at least mesenchymal stem cells from the collected body fluid. A method for producing a body tissue filling body, comprising: a step of extracting a body; a step of diluting the extracted body concentrate with a diluent; and a step of spraying the diluted body concentrate on a body tissue filling material. 2. The method for producing a biological tissue filling material according to claim 1, wherein the biological tissue filling material is processed according to the shape of the biological tissue defective portion before being sprayed on the biological tissue filling material. Method. 3. A method for producing a body tissue filling body for filling a body tissue defect portion of a patient during surgery, comprising a step of collecting a body fluid from the patient, and a concentrate containing mesenchymal stem cells from the collected body fluid. And a step of adhering the extracted concentrate to the body tissue filling body. 4. The method for producing a biological tissue filling material according to claim 3, wherein the biological tissue filling material is processed according to the shape of the biological tissue defective portion before the concentrated body is attached to the biological tissue filling material. 5. A body tissue filling body produced by the production method according to any one of claims 1 to 4 is kept at a predetermined temperature and humidity until transplanted into a body tissue defect portion of a patient. And a method for producing a moisturizing body tissue filler.