JP2006006546A - Bone prosthetic material, bone prosthesis and method of manufacturing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To increase the speed of absorption of a calcium phosphate porous material, to facilitate the formation of a bone matrix by osteoblasts, and to increase the speed of remodeling of a bone tissue, by making osteoclasts efficiently function. <P>SOLUTION: The bone prosthesis 1 comprises the calcium phosphate porous material 2 and a powder dentin material 3 included in the calcium phosphate porous material. The bone prosthesis has an effect of early restoration of a bone deficient part by activating osteoclasts, facilitating the absorption of the calcium phosphate porous material 2, and facilitating the metabolism. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

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

  In recent years, it has become possible to regenerate the bone and repair the defect by replenishing the bone defect caused by bone tumor extraction, trauma, etc. with a bone grafting material. Hydroxyapatite (HAP) and tricalcium phosphate (TCP) are known as bone prosthetic materials. From the idea that no foreign matter is left in the body, for example, a calcium phosphate porous material such as β-TCP is used. Scaffolding material is used. When β-TCP is brought into contact with bone cells in the bone defect portion, so-called remodeling is performed in which osteoclasts absorb β-TCP and osteoblasts form new bone. That is, the bone prosthetic material supplemented in the bone defect portion is replaced with autologous bone over time.

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 grafting material in order to increase the repair speed of a bone defect after surgery. The bone defect is filled with cultured bone containing many bone marrow mesenchymal cells that have been proliferated using the bone filling material as a scaffold by culturing, so compared to the method of growing cells in the body after surgery, The number of days until replacement can be greatly shortened (see, for example, 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, 30th Volume 10, No. 10, p. 46-49

  However, in order to further reduce the burden on the patient, it is necessary to further increase the repair speed of the postoperative bone defect, not only to activate the osteoblasts that newly form the bone matrix It is necessary to promote the metabolism by efficiently functioning or activating osteoclasts that absorb β-TCP.

  This invention has been made in view of the circumstances described above, and by efficiently functioning osteoclasts, the absorption of the calcium phosphate porous body is accelerated, and the formation of bone matrix by osteoblasts is promoted, An object of the present invention is to provide a bone filling material, a bone filling material, and a method for producing the same, which can improve the speed of remodeling of bone tissue.

In order to achieve the above object, the present invention provides the following means.
The present invention provides a bone grafting material comprising a calcium phosphate porous material containing a powdered dentin substance.
Since the dentin substance contains calcium phosphate, collagen protein, and metals such as Sr, Br, and Zn, it has an action of promoting the growth of osteoblasts and osteoclasts that come into contact therewith. Therefore, by seeding osteoblasts and osteoclasts on the bone grafting material of the present invention, cultured bone that is rapidly absorbed and rapidly forms a bone matrix can be produced. In addition, by directly filling the bone defect material of the present invention in the bone defect part in the living body, it is possible to promote the growth of surrounding osteoblasts and osteoclasts and to rapidly repair the bone defect part. It becomes. In this case, by forming the dentin substance in a powder form, it can be contained so as to be uniformly distributed in the bone grafting material, and uniform metabolism in each part can be achieved.

The present invention also provides a bone grafting material comprising a calcium phosphate porous body containing an osteoclast migration factor.
By filling the bone defect material according to the present invention in the bone defect part, osteoclast precursor cells existing in the living body can be attracted to the vicinity of the calcium phosphate porous body by the action of the osteoclast migration factor. Since osteoclast progenitor cells exist in the body, even if there is no osteoclast migration factor, it floats in the vicinity of the calcium phosphate porous body over time, but it is positively activated by the osteoclast migration factor. By attracting the target, the absorption of the porous calcium phosphate by the osteoclast can be started at an early stage. The induced osteoclast progenitor cells are differentiated into mature osteoclasts by the action of osteoclast differentiation factors supplied from osteoblasts existing in the body, and start to absorb calcium phosphate porous bodies. It becomes like this. As an osteoclast migration factor, for example, M-CSF (Macrophage-Colony
Stimulating Factor).

In the said invention, it is preferable to contain an osteoclast differentiation factor. Osteoclast differentiation factor is supplied from osteoblasts, but by containing calcium phosphate in advance, osteoclast progenitor cells can be turned into mature osteoclasts without waiting for supply from osteoblasts. It can be differentiated and absorption of the calcium phosphate porous material can be started at an early stage.
As an osteoclast differentiation factor, for example, sRANKL (soluble Receptor Activator of
Nuclear factor-kB Ligand
).

The present invention also provides a bone filling material comprising a calcium phosphate porous body containing osteoblasts and osteoclasts or precursor cells thereof.
According to the bone filling material according to the present invention, osteoclasts or their precursor cells that absorb calcium phosphate porous material and osteoblasts that form the bone matrix are preliminarily contained. By doing so, the resorption of calcium phosphate porous material by osteoclasts and the formation of bone matrix by osteoblasts is performed in a balanced manner, metabolism is promoted, and bone defects are replaced early to repair the defect. Can do. Since osteoblasts secrete osteoclast migration factor and osteoclast differentiation factor, differentiation into mature osteoclasts is promoted even when osteoclast progenitor cells are contained in advance. Bone defect can be repaired early.

The present invention also provides a bone filling material comprising a calcium phosphate porous body containing osteoblasts and an osteoclast migration factor.
According to the bone prosthesis according to the present invention, osteoclast progenitor cells existing in the body are attracted to the vicinity of the calcium phosphate porous material by the action of the osteoclast migration factor by filling the defect part of the bone tissue. can do. Thereby, absorption of the calcium-phosphate porous body by an osteoclast can be started at an early stage. The induced osteoclast precursor cells are differentiated into mature osteoclasts by the action of osteoclast differentiation factors supplied from osteoblasts contained in the calcium phosphate porous body, and the calcium phosphate porous body is absorbed. To start.

In the said invention, it is preferable to contain an osteoclast differentiation factor.
By containing osteoclast differentiation factor in advance, mature osteoclast progenitor cells that have been attracted by osteoclast migration factor without waiting for supply of osteoclast differentiation factor from osteoblasts. It can be differentiated into bone cells, and the absorption of the calcium phosphate porous material can be started at an early stage.

Moreover, in the said invention, it is preferable to contain a powdery dentin substance.
According to the present invention, the calcium phosphate porous body is rapidly absorbed by the osteoclasts activated by the dentin substance, and the bone matrix is rapidly formed by the osteoblasts activated by the dentin substance. The Thereby, it becomes possible to perform a quick repair of a bone defect part. In this case, by forming the dentin substance in a powder form, the dentin substance can be uniformly distributed in the bone filling body, and uniform metabolism in each part can be achieved.

  The present invention also includes a step of seeding and culturing a cell suspension containing mesenchymal stem cells in a calcium phosphate porous body, differentiating the mesenchymal stem cells into osteoblasts, and osteoclast together with powdered dentin material There is provided a method for producing a bone complement comprising the steps of culturing cells or progenitor cells thereof, and seeding the cultured osteoclasts on a calcium phosphate porous body containing osteoblasts.

  According to the present invention, mesenchymal stem cells seeded and cultured in a calcium phosphate porous body are differentiated into osteoblasts to produce bone matrix. On the other hand, osteoclasts or their progenitor cells cultured with a powdered dentin substance are proliferated by the action of the dentin substance and their functions are activated. Then, by seeding such osteoclasts or progenitor cells thereof into a calcium phosphate porous body containing osteoblasts, a bone filling body that starts absorption of calcium phosphate at an early stage is produced.

  ADVANTAGE OF THE INVENTION According to this invention, there exists an effect that osteoclast is activated, the absorption of a calcium-phosphate porous body is accelerated | stimulated, metabolism is promoted, and a bone defect part can be repaired at an early stage.

Hereinafter, a bone grafting material 1 according to an embodiment of the present invention will be described with reference to FIGS. 1 and 2.
The bone grafting material 1 according to the present embodiment has a structure in which a powdered dentin substance 3 is contained in a cubic block-shaped β-TCP porous body 2.

  Examples of the dentin substance 3 include ivory and teeth. The dentin material 3 contains hydroxyapatite as a main component and contains a small amount of a metal such as collagen protein and Sr, Br, Zn. The dentin substance 3 is pulverized so as to have an average particle diameter of about 100 μm to 1 mm, for example. The powdered dentin substance 3 can be produced, for example, by freeze pulverization or mechanical pulverization without applying heat that denatures the collagen protein contained therein.

  In order to manufacture the bone grafting material 1 according to the present embodiment, first, as shown in FIG. 2A, for example, a powdery β-TCP and a powdery powder are placed in a shallow container 4 A slurry 5 obtained by mixing a dentin substance and mixing it in a predetermined medium is poured to a predetermined depth and sintered. Thereby, as shown in FIG. 5B, a flat β-TCP porous body 6 is manufactured. Next, the bone of this embodiment is obtained by uniformly cutting powder dentin material 3 into a cubic block-shaped β-TCP porous body 2 as shown in FIG. The filling material 1 is obtained.

The operation of the bone grafting material 1 according to this embodiment configured as described above will be described below.
Since the bone prosthetic material 1 according to the present embodiment contains the dentin substance 3, cell adhesiveness is ensured by the hydroxyapatite that is a component of the dentin substance 3. In addition, the growth of cells is promoted by metals such as Sr, Br, Zn and the like contained in the dentin material 3 in a slight amount.

  That is, the bone grafting material 1 according to the present embodiment has an effect that cell proliferation can be promoted as compared with a bone grafting material made of a pure β-TCP porous body. Therefore, when the bone filling material 1 according to the present embodiment is directly filled in a bone defect part in a living body, osteoclasts and osteoblasts existing around the bone defect part are activated by the dentin substance 3. As a result, the resorption of the β-TCP porous body by osteoclasts is promoted, and the production of the bone matrix by the osteoblasts is promoted, so that the β-TCP porous body is quickly replaced with bone tissue. Become.

  In particular, since the dentin substance 3 is contained so as to uniformly disperse into the block-like β-TCP porous body 2, even for cells that have penetrated into the β-TCP porous body 2, The growth promoting effect by the dentin substance 3 can be achieved.

  In addition, although the bone grafting material 1 which concerns on this embodiment is manufactured by sintering the slurry which made the dentin substance 3 powdered and mixed with (beta) -TCP, it replaces with this and the dentin substance 3 is manufactured. Sintered slurry containing β-TCP containing no β-TCP to produce a β-TCP porous body 2, powdery dentin material 3 pulverized to have an average particle size of about 100 μm to 1 mm is obtained as β- The surface of the TCP porous body 2 may be sprinkled. Collagen protein, which is a component of dentin substance 3, is a component that constitutes an extracellular matrix produced by cells, and has the effect of increasing the adhesion of cells that come into contact therewith. By doing in this way, since the dentin substance 3 is not overheated, the bone grafting material 1 can be manufactured without denaturing the collagen protein which is one component of the dentin substance 3, and the action of increasing the cell adhesion by the collagen protein can be achieved. Can also be demonstrated.

Next, the bone grafting material according to the second embodiment of the present invention will be described.
In the following description, the same reference numerals are given to components common to the description of the first embodiment described above, and the description is omitted.
The bone grafting material according to the present embodiment is obtained by adding an osteoclast migration factor to the β-TCP porous body 2. As an osteoclast migration factor, M-CSF is mentioned, for example.
When the bone prosthetic material according to this embodiment configured as described above is compensated for in a bone defect part in a living body, the osteoclast precursor cells floating around the bone defect part by the action of osteoclast migration factor Has been attracted and adheres to the β-TCP porous body 2. Since osteoblasts are also present around the bone defect, when osteoblasts adhere to the β-TCP porous body 2 and produce bone matrix, osteoclast differentiation factor secreted simultaneously By the action, the osteoclast precursor cells that have been attracted are differentiated into mature osteoclasts, and the β-TCP porous body 2 is absorbed.

  That is, the bone defect portion is repaired by the absorption of the β-TCP porous body 2 by mature osteoclasts and the production of bone matrix by osteoblasts. In this case, according to the present embodiment, osteoclast progenitor cells are actively attracted to the β-TCP porous body 2 by the action of the osteoclast migration factor, so that the osteoclasts The start time of resorption by is accelerated, so that the early repair of the bone defect becomes possible.

Next, a bone grafting material according to a third embodiment of the present invention will be described.
The bone grafting material according to the present embodiment is obtained by further adding an osteoclast differentiation factor to the bone grafting material according to the second embodiment. Examples of osteoclast differentiation factors include sRANKL.
When the bone prosthetic material according to this embodiment configured as described above is compensated for in a bone defect part in a living body, the osteoclast precursor cells floating around the bone defect part by the action of osteoclast migration factor Has been attracted and adheres to the β-TCP porous body 2. The osteoclast progenitor cells that have been attracted are differentiated into mature osteoclasts by the action of osteoclast differentiation factors contained in the β-TCP porous body 2 and absorb the β-TCP porous body. It becomes like this.

  In other words, by waiting for the osteoclast differentiation factor to be secreted from osteoblasts by preliminarily containing an osteoclast differentiation factor that makes the osteoclast progenitor cells that have been attracted become mature osteoclasts. There is an advantage that the absorption of the β-TCP porous body 2 can be started earlier.

  In addition, in the bone grafting material which concerns on 2nd, 3rd embodiment, it is preferable to contain the dentin substance 3 similarly to the bone grafting material 1 which concerns on 1st Embodiment. The action of the osteoclast migration factor actively attracts osteoclast progenitor cells, and the action of the osteoclast differentiation factor promotes the formation of mature osteoclasts. In addition to accelerating the onset of resorption, activation of osteoblasts and mature osteoclasts by the action of the dentin substance 3 has the effect of promoting metabolism and rapid remodeling.

  Moreover, in the bone grafting materials according to the first to third embodiments, the cubic block-shaped β-TCP porous body 2 is adopted as the base material, but instead of this, the granular β-TCP porous body 2 is used. May be adopted. By doing in this way, it can adapt to the shape of a bone defect part flexibly, and can fill a bone defect part without gap.

Next, the bone graft according to the first embodiment of the present invention will be described with reference to FIG.
The bone filling material according to this embodiment includes osteoblasts and osteoclasts in the cubic block-shaped β-TCP porous body 2.

As the osteoblast, for example, a mesenchymal stem cell extracted from bone marrow fluid is primarily cultured to proliferate and then differentiated from a mesenchymal stem cell by adding a predetermined differentiation inducing factor and culturing is used. . It is effective to produce bone matrix to some extent by culturing osteoblasts seeded in the β-TCP porous body 2 for a predetermined period.
Osteoclasts are seeded from the patient's body. Since there are not many osteoclasts in the body, it is preferable to use those that have been cultured and proliferated by the following method.

  For example, as shown in FIG. 3, osteoclast culture is performed in a powdery dentin material having an average particle diameter of about 100 μm to 1 mm in each section of the petri dish 10 whose bottom surface is partitioned by a partition wall 10a. 3 is carried out in a state where a predetermined medium 11 is stored in the petri dish 10. As mentioned above, since the dentin substance 3 is mainly composed of hydroxyapatite, the cell adhesion is good, and the remaining collagen protein further improves the adhesion, so osteoclasts. Form an environment in which they can easily bond and grow easily. Moreover, since the dentin substance 3 contains some amount of metals such as Sr, Br, and Zn, it has an action of promoting the growth of osteoclasts. By culturing in this manner, osteoclasts are sufficiently proliferated.

  The partition wall 10a in the petri dish 10 is such that when the petri dish 10 is tilted or the like, the powdered dentin substance 3 remains in each section, and is not an essential configuration. Further, as shown in FIG. 4, a plate 12 made of β-TCP porous body 2 or hydroxyapatite may be disposed in the petri dish 10 and osteoclasts may be cultured on the plate 12. The plate 12 may be a flat plate or may be provided with a partition wall 12a similar to that shown in FIG.

  When it is desired to expand osteoclasts in a larger amount, as shown in FIG. 5, a powdered dentin substance 3 is filled in a cylindrical column 13, and dentinals are provided at the entrances on both sides thereof. You may decide to use the bioreactor 15 which has arrange | positioned the filter 14 which has a hole finer than the material 3 and larger than an osteoclast. Tubes 17 and 18 connected to the culture medium preparation tank 16 are connected to the inlet / outlet of the bioreactor 15, and a pump 19 is arranged in the middle of the inlet side tube 17. The osteoclasts collected from the patient are suspended in the culture medium 11 in the culture medium preparation tank 16, and the pump 19 is operated to circulate the culture medium 11, thereby bringing the osteoclasts together with the culture medium 11 into the bioreactor 15. Lead.

  Since the bioreactor 15 is filled with the dentin material 3, osteoclasts that have passed through the filter 14 and entered the bioreactor 15 adhere to the surface of the dentin material 3 and start growing. As described above, the dentin substance 3 has an osteoclast proliferation promoting action and is formed in a powder form, so that the culture area is expanded, so that a large amount of osteoclasts can be obtained. Since the filter 14 has a hole smaller than the dentin material 3, the filter 14 is held so that the dentin material 3 does not leak from the column 13.

  The obtained osteoclast may be seeded on the β-TCP porous body 2 in a form separated from the dentin material 3 by a proteolytic enzyme such as trypsin, or may be seeded together with the dentin material 3. . Seeding with the dentin substance 3 is preferable because the growth promoting effect of the dentin substance 3 promotes the growth of osteoclasts and osteoblasts. In this case, the dentin substance 3 can be killed by freeze-grinding, which is preferable in that a problem such as immune rejection does not occur.

According to the bone filling material according to the present embodiment configured as described above, the bone-removed portion is transplanted into the bone defect portion by the absorption of the β-TCP porous body 2 by osteoclasts and the production of bone matrix by osteoblasts Repair of the bone defect can be started at an early stage.
In the bone filling material according to the present embodiment, osteoclasts are seeded in the β-TCP porous body 2, but instead, osteoclast precursor cells may be seeded. . By seeding the progenitor cells, the progenitor cells are rapidly differentiated into mature osteoclasts by the action of osteoclast differentiation factor supplied from osteoblasts, and the resorption of β-TCP porous body 2 is started early. .

  In addition, osteoblasts obtained by differentiating mesenchymal stem cells extracted from bone marrow fluid by the culturing process are seeded in the β-TCP porous body 2, but instead, the mesenchymal stem cells are transformed into β-TCP porous. What is seeded on the body 2 may be cultured and differentiated into osteoblasts. Alternatively, instead of extracting mesenchymal stem cells, bone marrow fluid seeded as it is in the β-TCP porous body 2 may be cultured to form osteoblasts.

  Further, by inoculating osteoclasts or osteoclast precursor cells cultured with the dentin material 3 into the β-TCP porous body 2 together with the dentin material 3, cells by the components contained in the dentin material 3 are obtained. Although it is possible to utilize the growth promoting action, instead of the dentin substance 3, an alloy or metal ion containing a slight amount of a metal such as Sr, Br, Zn or the like is mixed alone into the β-TCP porous body. It may be.

It is the perspective view which fractured | ruptured one part explaining the bone grafting material which concerns on the 1st Embodiment of this invention. It is a figure explaining the manufacturing process which manufactures the bone grafting material of FIG. It is a figure explaining the culture container used for the osteoclast culture | cultivation process among the manufacturing methods of the bone filling body which concerns on the 1st Embodiment of this invention. FIG. 4 is a perspective view showing a modified example of FIG. 3 and a plate on which osteoclasts are placed and cultured. FIG. 9 is another example of the modification of FIG. 3 and is a diagram for explaining a method for culturing osteoclasts using a bioreactor.

Explanation of symbols

1 Bone prosthetic material 2 β-TCP porous material (calcium phosphate porous material)
3 Dentin substances

Claims (8)

  A bone filling material comprising a calcium phosphate porous material containing a powdered dentin substance.   A bone grafting material comprising osteoclast migration factor in a calcium phosphate porous material.   The bone grafting material according to claim 2, comprising an osteoclast differentiation factor.   A bone substitute comprising a calcium phosphate porous material containing osteoblasts and osteoclasts or precursor cells thereof.   A bone prosthesis comprising a calcium phosphate porous material containing osteoblasts and osteoclast migration factor.   The bone complement according to claim 5, which contains an osteoclast differentiation factor.   The bone filling material according to any one of claims 4 to 6, comprising a powdered dentin substance. Seeding and culturing a cell suspension containing mesenchymal stem cells on a calcium phosphate porous body, and differentiating mesenchymal stem cells into osteoblasts;
Culturing osteoclasts or precursors thereof with powdered dentin material;
A method for producing a bone filling material comprising the step of seeding cultured osteoclasts on a calcium phosphate porous material containing osteoblasts.

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