JP2006230817A - Biological tissue compensation material, and method of manufacturing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To grow cells from inside a biological compensation material while facilitating permeation of cells even into micropores and preventing the cells from easily flow out of the pores. <P>SOLUTION: The biological tissue compensation material introducing a platelet poor plasma into a porous material is provided. In addition, the biological tissue compensation material introducing a liquid including the platelet poor plasma and cells into the porous material in which the platelet poor plasma is gelated is provided. The method of manufacturing a biological tissue compensation body is provided by introducing a liquid including the platelet poor plasma, calcium chloride and cells into the porous biological tissue compensation material (S8) and gelating the liquid after the introduction. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a biological tissue filling material, a biological tissue filling material, and a method for producing the same.

  In recent years, in so-called regenerative medicine, mesenchymal stem cells are extracted from bone marrow cells or the like collected from a patient in order to increase the repair speed of a defect in a living tissue after surgery, and β tricalcium phosphate (β-TCP) or It has been proposed to produce a body tissue supplement represented by cultured bone by culturing with a body tissue filling material such as hydroxyapatite (HAP). Biological tissue replacements contain many mesenchymal stem cells that have already proliferated on a bone grafting material as a scaffold at the time of transplantation, so that after transplanting biomaterials that do not contain cells, the cells grow in the body to repair the tissue. Compared with the method to do, the days until it is replaced with a self-organized tissue can be significantly shortened (see, for example, Non-Patent Document 1).

  In order to produce a biological tissue complement, generally, first, mesenchymal stem cells taken out from the bone marrow cells of a patient are primarily cultured in a culture vessel to increase the number of cells to the required number. In this process, the grown cells are detached from the culture vessel at least once and transferred to a larger culture vessel. And when it finally increases to the required number of cells, it is peeled again from the culture vessel and attached to the biological tissue filling material, and the secondary culture is performed. Thereby, a biological tissue complement is manufactured (for example, refer nonpatent literature 2).

In this case, the mesenchymal stem cells are activated so that the formation of the living tissue is actively performed by adhering to the living tissue filling material and growing, so that the mesenchymal stem cell is used as the living tissue filling material. It is important that it adheres sufficiently. Conventionally, as a method for sufficiently attaching cells to a biological tissue filling material, it is possible to improve the material of the biological tissue filling material, to make it composed of a bioactive glass material or a bioactive ceramic material, and to treat it with an aqueous solution containing specific ions. It has been proposed (see, for example, 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, 30th Volume 10, No. 10, p. 46-49 Yoshikawa, “Cultivated dermis and bone marrow with bone marrow mesenchymal cells—Regenerative medicine with bone marrow mesenchymal cells”, Bioindustry, CMC Publishing Co., Ltd., 2001, Vol. 18, No. 7, p. 46-53 JP 2000-506738 A (page 10, lines 20-23)

  However, for example, a tissue filling material made of a material such as β-TCP reduces the porosity when filling a living tissue that needs to maintain high strength after regeneration, such as bone. Although it is necessary, there is a problem that the smaller the porosity, the more difficult the seeded cells enter the pores. That is, when the pores become small, it becomes difficult for the cells to enter the pores due to the viscosity and adhesiveness of the cells themselves. Therefore, for example, in order to make up a biological tissue defect portion of a predetermined size, a biological tissue filling material made of a relatively large block-like β-TCP porous body that matches the defect portion was seeded. It was difficult to infiltrate the cells into the living tissue filling material.

  On the other hand, in order to facilitate the entry of cells into the pores, it may be possible to reduce the viscosity of the liquid containing the cells. Before adhering to the inner wall, it also flows out of the living tissue filling material. It has been extremely difficult to adjust the viscosity of the liquid containing the cells so that the cells can easily enter the pores and hardly flow out.

  The present invention has been made in view of such circumstances, and while making it easy to permeate cells into fine pores, it is difficult to flow out of the pores, so that cells can be removed from the inside of the living tissue filling material. It aims at providing the manufacturing method of the biological tissue complement which can be made to grow.

In order to solve the above problems, the present invention provides the following means.
The present invention provides a biological tissue filling material obtained by introducing platelet-poor plasma into a porous material.
According to the present invention, platelet poor plasma (PPP) introduced into a porous material contains a material that gels with calcium chloride or the like such as sodium citrate. By introducing calcium or the like, PPP can be gelled and cells can be held in the pores of the porous material.

That is, when a liquid containing cells, calcium chloride, and the like is permeated into the pores of the biological tissue filling material, it can be suppressed to a relatively low viscosity. Therefore, at the time of introduction, penetration into the pores is promoted by a relatively high fluidity, and once introduced, the cells can be changed into a highly viscous gel to retain the cells in the pores. When cells are retained in the pores, they are retained in the PPP gel and stay inside the pores, or attach to the inner wall of the nearby pores, are activated, and fully differentiate into living tissues. Can do. As a result, even if the biological tissue filling material according to the present embodiment is in a relatively large block shape, a biological tissue filling material having cells that have grown sufficiently to the inside can be produced.
In addition, since PPP contains almost no platelet-derived growth factor that suppresses osteoblast differentiation, such as TGF-β and PDGF, it is possible to activate cells retained in the gel with very high efficiency. Become.

The present invention also provides a biological tissue filling material obtained by introducing a liquid containing PPP and cells into a porous material to gel the PPP.
According to the present invention, by virtue of the fact that the cell is activated by the three-dimensional structure of fibrin made from fibrinogen contained in the PPP, and the PPP does not contain a platelet-derived growth factor that suppresses cell differentiation, It becomes possible to activate cells retained in the gel with very high efficiency. Therefore, a defect part can be repaired at an early stage by transplanting to a biological tissue defect part or the like.

Moreover, this invention provides the manufacturing method of the biological tissue filling body which introduce | transduces the liquid containing PPP, calcium chloride, and a cell into a porous biological tissue filling material, and gelatinizes the said liquid after introduction | transduction.
According to the present invention, the liquid containing the cells is gelled after being introduced into the biological tissue filling material by the action of PPP and calcium chloride. Therefore, at the time of introduction into the biological tissue filling material, that is, the liquid containing cells. When it penetrates into the pores of the biological tissue filling material, it can be suppressed to a relatively low viscosity. Therefore, at the time of introduction, penetration into the pores is promoted by a relatively high fluidity, and once introduced, the cells can be changed into a highly viscous gel to retain the cells in the pores. When the cells are retained in the pores, they adhere to the inner wall of the nearby pores, are activated, and can fully differentiate into living tissues. As a result, even with a relatively large block-shaped biological tissue filling material, a biological tissue filling material having cells that have grown sufficiently to the inside can be produced.

  In the above-mentioned invention, it is preferable to add calcium chloride to a liquid containing cells and platelet-poor plasma before introducing the liquid into the body tissue filling material. In this way, when calcium chloride is added, using the property of the liquid containing PPP that gently starts to gel, the fluidity is maintained until the cells penetrate into the living tissue filling material, It becomes possible to retain the fluidity at an appropriate time when it reaches the pores.

  Moreover, in the said invention, it is good also as adding thrombin with calcium chloride and adjusting the quantity of the thrombin added according to the form of a biological tissue filling material. As a result, for example, for a tissue filling material with fine pores, the gelation rate can be slowed by reducing the amount of thrombin and slowing the coagulation reaction. For the material, the amount of thrombin can be increased to promote the coagulation reaction, and the gelation by the reaction between PPP and calcium chloride can be accelerated.

Moreover, in the said invention, it is preferable that the said cell is a cell cultured in the presence of dexamethasone.
According to the present invention, the ability to differentiate into progenitor cells such as osteoblasts is maintained by culturing cells from the initial culture stage in the presence of dexamethasone. Then, the cells cultured in the presence of dexamethasone are introduced into the biological tissue filling material together with the liquid containing PPP, and the cells are activated in the pores by gelling the PPP and staying in the pores of the biological tissue filling material. It is possible to promote the formation of a living tissue while maintaining the converted state.

  According to the present invention, it is possible to increase the permeability of cells into the biological tissue filling material and to retain the cells inside the biological tissue filling material, and to activate the cells inside the biological tissue filling material. It is possible to provide a biological tissue complement that has been grown to a high level.

Hereinafter, an embodiment of a biological tissue filling material, a biological tissue filling body, and a manufacturing method thereof according to the present invention will be described.
The biological tissue filling material according to the present embodiment is configured by introducing PPP into a porous material. The porous material is, for example, a block-shaped β-TCP porous body. PPP is obtained by centrifuging blood and removing blood cell components and platelets as much as possible.

  According to the biological tissue filling material according to the present embodiment configured as described above, when the cells are seeded on the biological tissue filling material, by adding calcium chloride in the cell suspension, In the pores of the material, gelation can be achieved by neutralizing sodium citrate, which is a chelating agent contained in PPP, with calcium chloride. Therefore, the cells contained in the gelled cell suspension are retained in the pores of the biological tissue filling material. As a result, the cells stay in the pores without flowing out of the living tissue filling material, and grow on the inner walls of the adjacent pores. Thereby, it becomes possible to manufacture the biological tissue filling body according to the present embodiment in a state where cells are also adhered to the inside of the biological tissue filling material.

  In PPP, cells are activated by the three-dimensional structure of fibrin made from fibrinogen. Furthermore, since the PPP does not contain a platelet-derived growth factor that suppresses cell differentiation, differentiation of the cells attached to the inner wall of the pores or retained in the gel is promoted, and the cells are high. It will be grown while maintaining activity. Therefore, after transplantation into the defect portion of the biological tissue, a biological tissue complement capable of repairing the defect portion at an early stage can be produced.

Next, the manufacturing method of the biological tissue filling body which concerns on one Embodiment of this invention is demonstrated below with reference to FIG. 1 and FIG.
In this production method, as shown in FIG. 1, first, for example, mesenchymal stem cells taken out from bone marrow cells are placed in a culture vessel together with a predetermined culture solution and cultured under a constant culture condition. (Step S1).

The culture solution is, for example, DMEM (Dulbecco's Modified Eagle's
Medium) and antibiotics mixed at an arbitrary blending ratio. The culture conditions are, for example, a temperature of 37 ° C. ± 0.5 ° C., a humidity of 100%, and a CO ₂ concentration of 5%.

  In this culturing process (step S1), the culture solution is exchanged periodically or as necessary (steps S2 and S3), and the culture vessel is changed to a larger one as the cells grow ( Steps S4 and S5).

  In addition, when the cells cultured while changing the culture container at least once are grown to the required number of cells (step S6), the cells are detached from the culture container and the connection between the cells is also disconnected. Are separated (step S7). Thereafter, the separated cells are mixed with a predetermined solution and then seeded on a biological tissue filling material composed of a β-TCP porous body block (step S8).

  In the figure, symbol S9 is a differentiation step, and a differentiation-inducing factor such as dexamethasone is added to mesenchymal stem cells proliferated to some extent. As a result, the mesenchymal stem cells are differentiated into a desired biological tissue, for example, osteoblast, and a biological tissue complement is produced. Differentiation step S9 is performed during culture before seeding of the cells on the biological tissue filling material as shown in FIG. 1, so that the cells induced to differentiate to some extent can be seeded on the biological tissue filling material. preferable. Moreover, code | symbol S10 is an evaluation step which evaluates the state of the cell contained in the manufactured biological tissue complement.

  In the seeding step S8, as shown in FIG. 2, the separated cells are first mixed with a culture solution (step S81), thereby forming a cell suspension having a predetermined concentration. The culture solution may be the same as the culture solution used for the culture described above. The cell suspension is set at a high concentration containing many cells in consideration of the amount of other gelable material such as PPP added thereafter. Next, PPP is added to the cell suspension (step S82). The amount of PPP added may be such that the fibrinogen concentration is approximately 2 to 45 mg / ml.

  Next, prior to the seeding of cells, a biological tissue filling material for seeding cells is prepared. The biological tissue filling material is, for example, a block-shaped β-TCP porous body. Then, a calcium chloride solution is added to the cell suspension containing PPP configured as described above (step S83). The calcium chloride solution to be added is obtained by dissolving calcium chloride so as to have a concentration of approximately 0.01 mol / l to 0.1 mol / l with respect to PPP.

  Since PPP contains sodium citrate, the added calcium chloride and sodium citrate react to initiate gelation. Since the gelation reaction is generally slow and takes several minutes for the viscosity to increase, immediately after adding calcium chloride, it is seeded on the biological tissue filling material by pipetting (step S84).

  If it is immediately after calcium chloride addition, gelation has not progressed and the viscosity of the cell suspension is kept low. Therefore, the cells can permeate into the pores of the tissue filling material relatively easily. When the cells penetrate to the center of the tissue filling material, the gelation reaction between sodium citrate and calcium chloride proceeds, the cell suspension is gelled, and the viscosity rapidly increases.

  As a result, the cells contained in the gelatinized cell suspension are retained in the pores of the biological tissue filling material. As a result, the cells stay in the pores without flowing out of the living tissue filling material, and grow on the inner walls of the adjacent pores. As a result, a body tissue filling body in which cells are also attached to the inside of the body tissue filling material is manufactured.

  As described above, according to the method for manufacturing a biological tissue filling body according to the present embodiment, in the infiltration stage in which cells penetrate into the pores of the biological tissue filling material, the pores are maintained by maintaining high fluidity of the cell suspension. Since the fluidity is lost by allowing the cell suspension to smoothly penetrate into the pores and gelling the cell suspension when the cells reach the inside of the pores, the cells can be easily attached to the inside of the tissue filling material. .

  In this case, according to the manufacturing method according to the present embodiment, since PPP is adopted as the gelable material, the action of the three-dimensional scaffold of fibrin formed by fibrinogen contained in PPP, Activation of the cells attached to the biological tissue filling material can be promoted. Therefore, it is possible to easily adhere to the inside of the pores and grow the cells three-dimensionally from the inside, and promote the activation of the cells to efficiently perform the tissue regeneration. Can be manufactured. Moreover, since PPP does not contain platelets or other substances that inhibit the activation of cells, the cells remaining in the gel can be maintained at a very high activity.

  Further, according to the manufacturing method according to the present embodiment, since the cells permeated into the pores are held in the pores by the gelled cell suspension, the cells are replaced with a living tissue filling material that has been conventionally required. There is also an effect that the culturing step for adhering to can be omitted. That is, since it takes time for the cells immediately after introduction to adhere to the pore inner wall of the biological tissue filling material, it is necessary to leave the cells in the culture vessel and to culture them for a predetermined time. However, the biological tissue complement produced by the production method according to the present embodiment is not required to stand because cells are prohibited from flowing out of the pores by the gelled cell suspension. Accordingly, the stationary culture step for attaching the cells to the biological tissue filling material can be omitted or shortened, so that the production efficiency can be increased and the production time can be shortened.

  Furthermore, since the cells of the biological tissue filling body produced by the production method according to the present embodiment continue to be retained in the pores, the cells are made of the biological tissue filling material before and after filling the biological tissue defect. Tissue regeneration can be performed efficiently without leaking outside.

  In this embodiment, a method of gelation by adding a calcium chloride solution to a solution obtained by adding PPP to a cell suspension is employed. By appropriately adjusting the amounts of thrombin and calcium chloride to be added, the degree of gelation, that is, the magnitude of the viscosity after gelation and the magnitude of the gelation speed can be adjusted. Moreover, blood components in the cell suspension are coagulated by fibrin formed by fibrinogen appropriately contained in the PPP, and the viscosity of the cell suspension is further increased.

  The amount of calcium chloride to be added and the appropriate amount of thrombin may be set according to the form of the biological tissue filling material to be seeded with cells. That is, it is considered that calcium chloride is increased so that gelation is started relatively early and a higher viscosity gel is formed for a living tissue filling material having large pores and cells easily flowing out. In addition, when the pores are fine and the viscosity is high, the cell suspension can hardly penetrate and the tissue filling material may have a relatively slow gelation rate and the final gel viscosity may be set low. . Therefore, in such a case, the amount of calcium chloride added may be small with the amount of thrombin contained in the PPP itself.

  Moreover, since PPP itself is collected from a patient, there are individual differences in gelation speed. Therefore, the amount of calcium chloride or the like to be added may be determined by component analysis of the collected PPP.

  In addition, the mesenchymal stem cells collected from bone marrow cells have been described as examples of cells to be seeded. However, the present invention is not limited to these, and ES cells, somatic stem cells, bone cells, chondrocytes, nerve cells, etc. Other somatic cells may be employed. In addition, although mesenchymal stem cells are collected from bone marrow, they may be collected from peripheral blood or umbilical cord blood instead.

  In addition, as a tissue replacement material for seeding cells, instead of β-TCP porous material, biocompatible porous ceramics, collagen, polylactic acid, polyglycolic acid, hyaluronic acid, or a combination thereof can be used. It may be used. Further, a metal such as titanium may be used. Moreover, you may employ | adopt arbitrary forms, such as a block form, a granule form, and a gel form.

[Example 1]
Next, an example of the biological tissue filling body and the manufacturing method thereof according to the above embodiment will be described below with reference to FIG.
First, a method for collecting PPP will be described.
Coagulation is prevented by collecting blood from Fisher rats and mixing with blood preservation solution (CPD solution) at the same time. The ratio of whole blood to CPD fluid is 10: 1. Whole blood was placed in a centrifuge tube and centrifuged at 312 G for 10 minutes, and the buffy coat and the upper layer were transferred to another centrifuge tube. This was further centrifuged at 1248G for 10 minutes, and the supernatant (9/10 amount) was collected. This is PPP. The remaining 1/10 is platelet rich plasma (PRP).

Next, the used cells will be described.
Mesenchymal stem cells collected from the femur bone marrow of Fischer rats are cultured at 37 ° C. ± 0.5 ° C., humidity 100%, CO ₂ concentration 5% in a culture solution in which DMEM and antibiotics are mixed at an arbitrary mixing ratio. After culturing for 10 days under the above culture conditions, dexamethasone, β-glycerophosphate and ascorbic acid phosphate were added to induce differentiation for 4 days.

Next, the biological tissue complement will be described.
The cultured cells are suspended in the above-described PPP and PRP, and a 2% calcium chloride solution (1/7 of the amount of PPP and PRP) is added, and immediately, a β-TCP porous body consisting of a cube having a side of 5 mm is formed. A biological tissue complement was produced by the introduction. The cell concentration is 2.6 × 10 ⁶ / ml. The produced biological tissue complement was transplanted subcutaneously into Fischer rats.

Next, the transplantation result will be described.
3 and 6 weeks after transplanting the biological tissue complement in which the PPP cell suspension and the PRP cell suspension were introduced into the β-TCP porous body, respectively, were collected after the passage of 3 weeks and 6 weeks. It was divided into 27 as shown. The results of analyzing the area ratio of new bone for the divided parts A to D are shown in FIG. 3 (b) and FIG. 3 (c). According to these, it can be seen that there are more new bones and the cell activity is higher in the case of PPP than in the case of PRP.

[Example 2]
Next, the effect of using PPP and dexamethasone together will be described.
Bone marrow fluid was collected from a Japanese monkey femur by puncture and cultured in a medium containing DMEM and FBS 10%. At this time, the cells were cultured with and without dexamethasone 10 nM. The bone marrow cells were cultured while being subcultured, and the obtained bone marrow cells were seeded in a β-TCP porous body.

  The sample was cultured without adding dexamethasone, and the bone marrow cell suspension obtained by adding dexamethasone, β-glycerophosphate, and ascorbic acid phosphate immediately before transplantation (4 days before transplantation) was negative-pressured and β-TCP porous. Cell suspension of bone marrow cells obtained by adding dexamethasone to the body (DEX-), adding dexamethasone from the beginning of culture, and adding dexamethasone, β-glycerophosphate, ascorbic acid phosphate immediately before transplantation (4 days before transplantation) The solution was introduced into the β-TCP porous body under negative pressure (DEX + PPP-), and the bone marrow cells cultured with dexamethasone added from the beginning of the culture were introduced into the β-TCP porous body for gelation. (DEX + PPP +) was prepared. These samples were transplanted subcutaneously in the back of the Japanese macaque, collected 5 weeks after transplantation, and observed with a microscope.

The results are shown in FIGS. 4 to 6, (a) shows a 1.25-fold magnified photograph, and (b) shows a 10-fold magnified photograph.
4 to 6, it can be seen that the bone tissue X spreads over the entire area of the β-TCP porous body in the order of (DEX−), (DEX + PPP−), and (DEX + PPP +). From this, it was shown that the biological tissue complement to which dexamethasone was added from the beginning of the culture and PPP was introduced was effective for bone formation.

It is a flowchart which shows the manufacturing method of the biological tissue complement body which concerns on one Embodiment of this invention. It is a flowchart explaining the cell seed | inoculation step in the manufacturing method of FIG. 1 shows Example 1 of a biological tissue filling body according to an embodiment of the present invention, (a) is a schematic diagram showing the position of a sample, (b) is a new bone area ratio for each sample after three weeks, PRP The graph shown in contrast with the contained biological tissue complement, (c) is a graph showing the ratio of new bone area for each sample after the lapse of 6 weeks in comparison with the biological tissue supplement containing PRP. It is a microscope picture which shows the formation state of the bone tissue at the time of using the cell cultured without adding dexamethasone as a comparative example of the biological tissue complementation which concerns on one Embodiment of this invention (it does not use PPP). It is a microscope picture which shows the formation state of the bone tissue at the time of using the cell cultured in the presence of dexamethasone as a comparative example of the biological tissue complement according to one embodiment of the present invention. It is a microscope picture which shows Example 2 of the biological tissue filling body which concerns on one Embodiment of this invention, and shows the formation state of the bone tissue at the time of using the cell cultured in the presence of dexamethasone with PPP.

Explanation of symbols

S8 seeding step S82 PPP addition step S83 calcium chloride addition step S84 cell seeding step

Claims (6)

  A tissue filling material obtained by introducing platelet-poor plasma into a porous material.   A biological tissue filling material obtained by introducing a platelet-containing plasma and a liquid containing cells into a porous material to gel the platelet-free plasma.   A method for producing a biological tissue filling material, wherein a liquid containing platelet-poor plasma, calcium chloride and cells is introduced into a porous biological tissue filling material, and the liquid is gelled after the introduction.   The method for producing a body tissue complement according to claim 3, wherein calcium chloride is added to a fluid containing cells and platelet-poor plasma before introducing the fluid into the body tissue filling material. Add thrombin with calcium chloride,
The manufacturing method of the biological tissue filling body of Claim 4 which adjusts the quantity of the thrombin added according to the form of the biological tissue filling material.   The method for producing a biological tissue complement according to any one of claims 3 to 5, wherein the cells are cells cultured in the presence of dexamethasone.

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