JP2008289734A - Creation method for osteoblast-base material complex - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a creation method for an osteoblast-base material complex excellent in its affinity for a living body. <P>SOLUTION: A method for cultivating an osteoblast is provided, the method using a cell culture base material comprising metal nonwoven fiber and using a process for seeding the osteoblast on the base material comprising the metal nonwoven fiber and performing gyratory culture of the base material where the osteoblast has been seeded. By the method, the osteoblast-base material complex can be easily manufactured. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing an osteoblast-substrate complex.

  In the past, in order to make up for or regenerate bone defects, various artificial implants have been developed in addition to transplanting bones from other parts of the body. In particular, a complex in which an artificial material such as hydroxyapatite or β-TCP which is a bioabsorbable ceramic is used as a cell culture substrate (scaffold) and stem cells are added to the substrate is representative.

  However, these materials also have problems in the treatment period and durability, and are not suitable for long-term use. Therefore, titanium or a titanium alloy has been developed as a base material having a strength higher than that of living bone and capable of shortening the healing period (see, for example, Patent Documents 1 to 4).

Special table 2003-533276 JP2004-16398 Special table 2004-53461 JP-A-2005-329060

  However, when titanium non-woven fabric is used as the cell base material, the cells are unevenly fixed on the non-woven fabric, or the cells stay outside the non-woven fabric and do not enter the inside. -When the substrate composite was transplanted, the affinity with the living body was poor.

  Therefore, an object of the present invention is to provide a method for producing an osteoblast-substrate complex having a better affinity with a living body.

  The present inventors use a base material made of a metal non-woven fiber as a scaffold for osteoblasts, and the osteoblasts uniformly enter the base material by swirling and stirring the base material seeded with osteoblasts. And found to proliferate efficiently.

That is, the present invention is as follows.
(1) A method for producing an osteoblast-cell culture substrate complex, the step of seeding osteoblasts on a cell culture substrate composed of a metallic nonwoven fiber, and the substrate on which osteoblasts are seeded And swirling culture of the material.

  (2) The method according to (1), further comprising a step of inducing mesenchymal stem cells into osteoblasts.

  (3) The method according to (1) or (2), wherein the osteoblast is derived from a human.

  (4) The method according to any one of (1) to (3), wherein the metal is titanium or a titanium alloy, or is titanium-coated.

  (5) An osteoblast-substrate complex produced by the method according to any one of (1) to (4).

  (6) A method for treating or supplementing bone defects in humans or non-human vertebrates, wherein vertebrate osteoblasts are seeded on a cell culture substrate made of a metallic nonwoven fiber; A method comprising swirling the cultured substrate and transplanting the swirled substrate into a vertebrate.

  (7) The method according to (6), comprising a step of collecting osteoblasts from a human or a non-human vertebrate.

  (8) The method according to (6), further comprising a step of collecting mesenchymal stem cells from a human or a non-human vertebrate, and a step of inducing the mesenchymal stem cells into osteoblasts.

  According to the present invention, it is possible to provide a method for producing an osteoblast-substrate complex having better affinity with a living body.

  Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Osteoblast-substrate complex and method of use thereof ==
The osteoblast-substrate complex according to the present invention is a base material on which cells are seeded by seeding osteoblasts on a base material using a cell culture base material and osteoblasts made of a non-woven fiber made of metal. Can be produced by swirling culture.

  Although it does not specifically limit as a metal material of the metal nonwoven fabric fiber used for a base material, Especially titanium (a titanium oxide is included) or a titanium alloy is preferable. Alternatively, a metal such as stainless steel may be coated with titanium, in which case the coating method is not particularly limited. The diameter of the fiber is not limited, but is preferably 10 to 200 μm. In addition, the density of the fiber in the cell culture substrate is not limited as long as the air permeability required for culturing the cells is ensured, but considering the durability of the substrate, the porosity is 40 to 95%. Preferably, 60 to 80% is more preferable, and 85 to 90% is particularly preferable. Furthermore, a completely communicating hole structure is preferable. In addition, the shape of the cell culture substrate made of a metallic non-woven fiber is not particularly limited, such as a disk shape (disk shape) or a rectangular parallelepiped shape.

  Osteoblasts used for culture can be collected by bone marrow puncture from bone marrows such as jaw bones and femurs of mice and humans. Alternatively, mesenchymal stem cells may be collected from the bone marrow and cultured to induce differentiation into osteoblasts. For differentiation induction into osteoblasts, a known method such as using a differentiation induction medium can be used.

  The medium used for the swirl culture is not particularly limited. However, in order to apply pressure in the differentiation direction from the mesenchymal stem cells to the osteoblasts and from the osteoblasts to the bone cells even during the swirl culture, Preferably it is done. Although the culture conditions are not particularly limited, the culture temperature is preferably 37 ° C., and the culture time is preferably 1 day to 8 weeks, particularly preferably 1 week to 6 weeks. The turning speed is not particularly limited, but is preferably 40 to 100 rpm, and more preferably 60 to 80 rpm. It is to be noted that the cells may be adhered to the bottom surface of the substrate by placing the cell-seeded substrate on a net or the like and reducing the contact of the bottom surface during swirling culture.

  The thus produced osteoblast-substrate complex can be used to treat or compensate for bone defects, for example, defects such as joints, tooth roots, skulls, or long bones.

  At this time, it is preferable to mold the base material in accordance with the shape of the bone defect before seeding the osteoblasts on the base material. The shape of the bone defect can be measured by a conventional method such as X-ray or CT. In order to mold the base material, for example, a ceramic mold having the shape of the measured defect portion is prepared, and the base material having the shape of the bone defect portion is obtained by filling the die with the base material and firing it. .

  Moreover, you may treat or supplement the target bone defect part by combining a base material and another support | carrier. The carrier used here is not particularly limited. For example, after fixing a cell culture substrate around a known implant body made of ceramic or hydroxyapatite, seeding osteoblasts and swirling culture An implant body provided with the osteoblast-substrate complex according to the present invention can be obtained.

== How to treat or compensate for bone defects in humans and non-human vertebrates ==
A bone defect can be treated or compensated by transplanting the osteoblast-substrate complex produced by the above-described method into a bone defect part of a human or a non-human vertebrate. As shown in Examples, by producing an osteoblast-substrate complex by the above-described method, osteoblasts uniformly invade into the substrate and efficiently proliferate.

  In this case, as osteoblasts to be seeded on the cell culture substrate, osteoblasts collected from vertebrates, or preferably those obtained by inducing mesenchymal stem cells collected from vertebrates into osteoblasts are used. In addition, it is preferable in terms of immunology to collect osteoblasts or mesenchymal stem cells from the same individual to which the osteoblast-substrate complex is transplanted.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.

[Example 1] Effect of culture method on bone formation ability In this example, a cell culture substrate composed of a metallic nonwoven fiber was used, and mesenchymal stem cells were cultured while being differentiated into osteoblasts. The results were compared with those during static culture.

1. Mesenchymal Stem Cells and Cell Culture Substrates First, mesenchymal stem cells collected by bone marrow puncture from the femur of Fisher344 rats (7 weeks old, male) were collected using a T-225 flask with 15% fetal bovine serum ( Primary culture was performed in a minimum essential medium (MEM) containing Fetal Bovine Serum (FBS).
Pure titanium non-woven fiber formed into a disk shape to be used as a cell culture substrate (Titanium-Web (TW), fiber diameter 50 μm, disk diameter 5 mm x thickness 2 mm, completely communicating hole structure, porosity 87%) (Manufactured by Hyrex Corporation) was previously degreased and further sterilized by dry heat at 190 ° C. for 6 hours immediately before the seeded cells.

2. Culture conditions 2-1. Static culture A mesenchymal stem cell solution was suspended at a concentration of 2.5 × 10 ⁶ cells / mL in osteoblast differentiation medium (MEM containing 10 nM dexamethasone, 0.28 mM ascorbic acid and 10 mM β-glycerophosphate). Degreased and sterilized TW was added to a 96-well plate at 1 disk / well, a cell suspension was added at 200 μL / well (5 × 10 ⁵ cells / well), and the cells were seeded. After pipetting three times, stationary culture was performed at 37 ° C.

2-2. Swirl culture After static culture overnight by the above method, a pure titanium wire mesh was laid under the TW. Subsequently, horizontal swirling culture was performed at 37 ° C. and 70 rpm (THE BELLY BUTTON, Stoval Life Science, Inc., USA). The rotation was continued during the culture except for the medium exchange.

3. Comparison between swirl culture group and stationary culture group On the 14th day after the start of culture, live cells were stained for each of stationary culture and swirl culture using Live / dead Viability Assay Kit (Molecular Probe), and fluorescent. Observed with a microscope. The results are shown in FIG.

  In addition, on the 7th and 14th days after the start of the culture, the cultured substrate is placed in a 2.0 mL microtube together with a predetermined amount of 10 mM Tris-HCl and 1 mM EDTA (pH 7.4), and the cells and the substrate. The material was crushed. The amount of DNA, the activity of alkaline phosphotase (Alkaline Phosphatase, ALPase), which is an osteoblast marker, and the amount of calcium were measured for the obtained specimens as follows. The measurement results are shown in FIGS.

  The method for measuring the amount of DNA is as follows. First, 200 μL of Hoechst 33258 (molecular probe) (5 μg / mL) -containing buffer was added to 20 μL of the supernatant in the sample, and the fluorescence after 5 minutes was measured with a plate reader (Wallac 1420 ARVOsx; PerkinElmaer Life and Analytical Sciences, Boston, MA). Salmon sperm DNA (Invitrogen) was used as the DNA standard solution.

  In measuring ALPase activity, 20 μL of the supernatant and 180 μL of pNPP (p-nitrophenyl phosphate) solution (Zymed Laboratories, South San Francisco, Calif.) Were added to a transparent plate and incubated at 37 ° C. for 30 minutes. Since pNPP is reduced to pNP in the presence of ALPase activity and exhibits a yellow color, the change in absorbance due to this coloration was measured using a plate reader. For control, a pNP solution was used.

  In measuring the calcium amount, 500 μL of 20% formic acid was added to the remaining sample to form a 10% formic acid solution, and the sample after decalcification at 4 ° C. for 3 days or more was used as the calcium measurement sample. This specimen was diluted with pure water and measured by the ICP method (SPS7800 plasma spectrometer; Seiko Instruments Inc., Chiba, Japan).

4). Results As observed in FIG. 1, in swirl culture, cells infiltrated into the base material along the titanium fiber and proliferated, and swirl culture has higher affinity between titanium and cells. It has been shown. In addition, cell culture on the bottom surface of the substrate has become possible by culturing on pure titanium wire mesh during swirl culture and making point contact with the bottom surface of the container.
In the measurement of the amount of DNA shown in FIG. 2, the swirl culture group showed very good cell growth compared to the stationary culture group. From the measurement of ALPase activity shown in FIG. 3, the swirl culture group showed very good osteoblast differentiation induction compared to the stationary culture group. Furthermore, the measurement of the amount of calcium shown in FIG. 4 also showed that osteoblasts were significantly ossified in the swirl culture group compared to the stationary culture group.

  Thus, swiveling culture of osteoblasts using T-W as a base material is a very simple and high-performance culture method even in view of clinical application.

[Example 2] Effect of swirling culture on bone formation ability When culturing mesenchymal stem cells, differentiation induction into osteoblasts was previously induced, seeded on a substrate, and then subjected to horizontal swirling culture (Osteoblast Like Cells, OBSc. And abbreviated as OBSc group). The results were compared with those obtained by culturing mesenchymal stem cells not previously induced in osteoblasts using a substrate (Mesenchymal Stem Cells, MSCs; hereinafter abbreviated as MSCs group).

1. Culture 1-1. MSCs group By the method described in Example 1, mesenchymal stem cells obtained from Fisher344 rat-derived femurs were primarily cultured in MEM containing 15% FBS.
1-2. OBSc group The mesenchymal stem cells obtained from the Fisher344 rat-derived femur used in the MSCs group were cultured in an osteoblast differentiation induction medium for 7 days, and the mesenchymal stem cells were induced to differentiate into osteoblasts.

2. The MSCs group or OBSc group was suspended in a main culture osteoblast differentiation induction medium at a concentration of 2.5 × 10 ⁶ cells / mL. Degreased and sterilized TW was added to a 24-well plate at 1 disk / well, and each cell suspension was seeded at 200 μL / well (5 × 10 ⁵ cells / well). After pipetting three times, static culture was performed overnight at 37 ° C. A pure titanium wire mesh was laid under TW, followed by horizontal swirling culture at 70 rpm (THE BELLY BUTTON, Stoval Life Science, Inc., USA).

3. Comparison of culture results At the time of 1st, 7th and 14th after the swirling culture, the measurement of live cell staining, DNA amount, ALPase activity, and calcium amount was performed in the same manner as in Example 1.
In measuring the amount of osteocalcin, the solution after the calcium amount measurement was centrifuged, and 500 μL of the supernatant was passed through a NAP-5 column (Sephadex G-25 DNA grade, Amersham Bioscience, Uppsala, Sweden), and further 10% formic acid 1.0 mL was passed through the column to recover a 1.5 mL solution. This solution was centrifuged, and the precipitate resuspended in 500 μL of buffer was used as an osteocalcin measurement sample. The amount of osteocalcin synthesized was quantified using a rat osteocalcin EIA kit (No. BT-490; Biomedical Technologies Inc., MA, USA).

  The results of live cell staining are shown in FIG. Moreover, the measurement result of DNA amount, ALPase activity, calcium amount, and osteocalcin amount is shown in FIGS.

4). Results From FIG. 5, in the MSCs group, the cells proliferated unevenly so as to stick to the outside of the substrate, whereas in the OBCs group, the cells entered the substrate and adhered along each fiber. However, it was growing uniformly. From this, it is considered that the OBCs group has better affinity between the cell-substrate complex and the living body than the MSCs group.
From FIG. 6 and FIG. 7, although the cell growth is more active in the MSCs group, the level of differentiation marker of osteoblasts is higher in the OBCs group in the initial stage (1st day and 7th day), and in the 14th day In the OBCs group, the level of osteoblast differentiation marker is lower. This is probably because osteoblasts differentiated into bone (bone formation) from day 7 in the OBCs group, and the level of osteoblast differentiation markers decreased.
From FIG. 8 and FIG. 9, it is considered that both the amount of calcium and the amount of osteocalcin are more in the OBCs group than in the MSCs group, and the bone formation of the cells is more advanced.
In this way, the OBCs group has better affinity between the cells and the base material, the cells grow uniformly, and the degree of bone formation is higher than the MSCs group. It was.

5. Transplantation of osteoblast-substrate complex in vivo Further, these cultured osteoblast-substrate complexes were implanted subcutaneously into the back of syngeneic rats. Three and six weeks after transplantation, the in vivo bone forming ability was evaluated by ALPase activity, calcium content, osteocalcin content, and image analysis by μCT.
FIG. 10 shows the ALPase activity measurement, calcium content measurement result, and osteocalcin content measurement result in vivo, and FIG. 11 shows the image analysis result by μCT.

6). Results FIG. 10 shows that the MSCs group has more osteoblasts, and the OBCs group has more bone formation.
In addition, FIG. 11 shows that in the OBCs group, a large amount of bone tissue is uniformly generated inside the base material.
Thus, after pre-inducing mesenchymal stem cells into osteoblasts during the initial culture, the osteoblast-substrate complex seeded on TW and cultured in a swivel manner is very excellent in bone formation in vivo after transplantation. And showed uniform bone tissue formation inside the substrate.

The result of Live / Dead dyeing | staining of the stationary culture group and swirl culture group in one Example is shown. The result of the DNA amount measurement of the stationary culture group and the swirl culture group in one Example is shown. The result of the ALPase activity measurement of the stationary culture group and the swirl culture group in one Example is shown. The result of the calcium content measurement of the stationary culture group and swirl culture group in one Example is shown. The result of the living cell dyeing | staining of the MSCs group and OBCs group in one Example is shown. The result of the DNA amount measurement of the MSCs group and OBCs group in one Example is shown. The result of the ALPase activity measurement of the MSCs group and OBCs group in one Example is shown. The result of the calcium content measurement of the MSCs group and OBCs group in one Example is shown. The result of the osteocalcin amount measurement of the MSCs group and OBCs group in one Example is shown. The results of in vivo ALPase activity, calcium content, and osteocalcin content of an osteoblast-substrate complex in one example are shown. The result of the in-vivo image analysis of the osteoblast cell-base_material composite_body | complex in one Example is shown.

Claims (6)

A method for producing an osteoblast-substrate complex comprising:
Seeding osteoblasts on a substrate composed of a metallic nonwoven fiber;
Swirling culture of the substrate seeded with osteoblasts;
A method comprising the steps of:   The method according to claim 1, further comprising the step of inducing mesenchymal stem cells into osteoblasts.   The method according to claim 1 or 2, wherein the osteoblast is derived from a human.   The method according to claim 1, wherein the metal is titanium or a titanium alloy, or is titanium-coated.   An osteoblast-substrate complex produced by the method according to any one of claims 1 to 4.   An osteogenesis promoting material containing the osteoblast-substrate complex according to claim 5.