JP2001103964A - Method for inhibiting osteoclast formation or method for inhibiting bone resorption - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for inhibiting osteoclast formation or a method for inhibiting bone resorption both by physical and uninvasive ultrasonic wave stimulation where the danger of side effect is low. SOLUTION: The method for inhibiting osteoclast formation or the method for inhibiting bone resorption is conducted under exposure to ultrasonic wave of a culture system containing osteoclast progenitor cells and inducers in the culture solution. The method for inhibiting osteoclast formation or the method for inhibiting bone resorption may be conducted under exposure to ultrasonic wave of a cocultivation system containing osteoclast progenitor cells in the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for suppressing osteoclast formation or a method for suppressing bone resorption, which is characterized by irradiating ultrasonic waves (hereinafter sometimes referred to as "US"). A method for inhibiting cell formation or a method for inhibiting bone resorption.

[0002]

Conventionally, as a method of inhibiting osteoclast formation or bone resorption, for example, a method of adding bisphosphonates to a co-culture system has been known [Bilezikia
n JP, Raisz LG, Rodan GA eds.Principales of bone
biology Academic Press, New York (1996)]. Clinically, drugs that inhibit osteoclast formation or bone resorption have been used for the treatment of bone-related diseases based on bone resorption by osteoclasts [Bilezikian JP, Raisz LG, Rodan GA eds. Princip
ales of bone biology Academic Press, NewYork (199
6)]. However, the method of adding or administering these drugs or pharmaceuticals has been problematic in that they are taken directly into the body or into cells and always involve the risk of side effects. Therefore, a method of inhibiting osteoclast formation or inhibiting bone resorption with a low risk of such side effects has been required.

[0003]

The inventors of the present invention have conducted intensive studies to find a method of inhibiting osteoclast formation or bone resorption with less risk of such side effects. The inventor has found that invasive ultrasonic stimulation can suppress osteoclast formation or bone resorption, and arrived at the present invention.

[0004]

That is, the present invention provides a method for inhibiting osteoclast formation, which comprises irradiating a culture system containing an osteoclast precursor cell and an inducing factor in a culture solution with ultrasonic waves. Alternatively, it is a method for inhibiting bone resorption, or a method for inhibiting osteoclast formation or inhibiting bone resorption, which comprises irradiating an ultrasonic wave in a co-culture system containing osteoclast precursor cells in a culture solution.

Hereinafter, the present invention will be described in detail. The osteoclast precursor cells used in the present invention include those in which osteoclasts are formed by inducing factors contained in the culture solution, or other support cells having the ability to support osteoclast formation in the culture solution. Osteoclasts are formed by coexistence. As osteoclast precursor cells,
Examples include bone marrow-derived progenitor cells, spleen-derived progenitor cells, peripheral blood monocyte-derived progenitor cells, and the like. The cells can be obtained from bone marrow, spleen, or peripheral blood of humans, mice, and the like, and can be obtained by culturing and subculturing using a medium for culturing osteoclast precursor cells.

Here, as inducers that act on osteoclast precursor cells to form osteoclasts, macrophage colony stimulating factor (hereinafter referred to as “M-CSF”), osteoclast-forming factor (RANKL / TRANCE / ODF / OPGL, hereafter "RANKL"
That. ), Tumor necrosis factor (hereinafter referred to as "TNF"), which is at least one selected from interleukin-4 (IL-4) and vascular endothelial cell growth factor (VEGF). May be used. Among these, it is preferable to use RANKL, and it is particularly preferable to use artificially produced soluble RANKL at a concentration of 1 to 250 ng / ml. These inducers are added to the culture solution immediately after seeding the osteoclast precursor cells until the osteoclasts are formed.

The cells other than the osteoclast precursor cells contained in the co-culture system include at least one selected from osteoblasts, stromal cells, fibroblasts, T cells, and B cells. Only one type may be used, or several types may be used.

Further, it is preferable that the culture solution of the co-culture system contains an inducing factor. Examples of the inducers used here include M-CSF, RANKL, TNF, IL-4, and VEGF, as well as interleukin-1 (hereinafter, referred to as “IL-1”).
Interleukin-3 (hereinafter referred to as “IL-3”), Interleukin-6 (hereinafter referred to as “IL-6”), Interleukin-11 (hereinafter referred to as “IL-11”), Interleukin-15 ( Prostaglandins such as interleukin-17 (hereinafter referred to as "IL-17"), prostaglandin E2 (hereinafter referred to as "PG"), and parathyroid hormone (hereinafter referred to as "IL-15"). PTH), parathyroid hormone-related peptides (hereinafter “PTHr
P ". ), Granulocyte / macrophage colony stimulating factor (hereinafter referred to as “GM-CSF”), and active vitamin Ds such as 1α, 25-dihydroxyvitamin D3 (hereinafter referred to as “VD”). At least one or several types selected can be used.

Among the above inducers, it is particularly preferred to use RANKL. This is because RANKL is an essential factor for osteoclastogenesis in vivo and plays an important role in bone metabolism and maintenance [Takahashi N, et al. Biochem.
Biophys Res Comm 256: 449-455 (1999)].
RANKL is present on cells that support osteoclast formation in vivo, such as osteoblasts, stromal cells, fibroblasts, etc., and is a receptor (RANK / TRANC) of osteoclast precursor cells.
ER / ODFR) to induce osteoclastogenesis [Takahashi N, et al. Biochem Biophys Res Com
m 256: 449-455 (1999)]. Since RANKL does not exist as a soluble factor in the living body, the osteoclast-forming system by co-culture is considered to be an evaluation system closer to the in vivo environment. RANKL plays an important role in normal bone metabolism, but is also deeply involved in bone-related diseases. For example, in postmenopausal osteoporosis, the number and bone resorption activity of osteoclasts increase and bone mass decreases, but it is thought that the formation of osteoclasts at this time is related to an increase in RANKL [ Kong YY, et al. Nature 397: 315-323 (199
9)]. Osteoclastogenesis inhibitor (hereinafter referred to as “OPG / OCIF”), which is an in vivo molecule that prevents RANKL from binding to RANKL,
In addition to inducing osteoclast formation induced by RANKL, animal models of bone-related diseases based on increased osteoclast formation and increased osteoclast activity, such as hypercalcemia and osteoporosis models, It has been shown that by suppressing bone cell formation and suppressing bone resorption activity, symptoms such as decreased bone mass and increased serum calcium levels are improved [Lacey DL, et al. Cell
93: 165-176 (1998)]. Thus, RANKL or RAN
Stimulation of osteoclasts caused by the involvement of factors that induce KL, as well as bone-related diseases that are thought to promote bone resorption, such as osteoporosis, bone loss or destruction of rheumatoid arthritis, artificial joints For bone loss immediately after replacement, loosening after artificial joint replacement, bone destruction due to bone metastases, etc., induction of osteoclasts or suppression of bone resorption by RANKL is a treatment method for the bone-related disease. Application is expected.

[0010] The co-culture system in the present invention refers to a method of culturing osteoclast precursor cells and at least one cell having the ability to support osteoclast formation in the same medium. here,
Cells having the ability to support osteoclast formation include osteoblasts,
At least one selected from stromal cells, fibroblasts, T cells, and B cells can be mentioned, and these may be used alone or several types may be used. Among them, it is preferable to use stromal cells, and it is more preferable to use bone marrow-derived stromal cells. As the combination with osteoclast precursor cells, it is most preferable to combine bone marrow-derived osteoclast precursor cells and bone marrow-derived stromal cells. Examples of bone marrow-derived stromal cells include mouse bone marrow stromal cells such as ST-2 and TSB-13-9. The ST-2 can be obtained from the American Type Culture Collection, and it can be cultured and subcultured in a medium described in detail below and used for experiments.

The culture solution for culturing osteoclast precursor cells used in the present invention contains 8-1000 ng / ml of M-CSF and contains 5%
Alpha MEM medium to which 15% fetal bovine serum is added. Among them, it is preferable to use an alpha MEM medium containing 100 ng / ml M-CSF and supplemented with 10% fetal bovine serum.

[0012] As the culture solution used in the co-culture system,
The induction factors, include between 5% and 15% fetal bovine serum, etc. alpha MEM medium or the like by adding, in particular, 1 × 10 ^-10 M~1 × 10
It is preferable to use an alpha MEM medium supplemented with 1α, 25-dihydroxyvitamin D3 in the range of ⁻⁷ M and 10% fetal bovine serum. As a time to add these inducers to the medium, the osteoclast precursor cells and cells having the ability to support osteoclast formation such as bone marrow-derived stromal cells are simultaneously rolled up,
It can be added until osteoclasts are formed.

The ultrasonic wave applied in the present invention means a low-output ultrasonic wave that does not generate heat. Specifically, a frequency of 1.3 to 2 MHz, a repetition frequency of 100 to 1000 kHz, and a burst width of 10
It is a low-power ultrasonic wave having an output of 100 mW / cm ² or less for 2000 μsec. As a method of irradiating the ultrasonic waves, for example, a low-output ultrasonic pulse is transmitted to the bottom of the culture well using an ultrasonic output unit of Exogen for 20 minutes a day, and is continuously irradiated for 3 to 5 days. Irradiation conditions include, for example, as a characteristic of ultrasonic output, a pulse width of 200 μsec, a frequency of 1.5 MHz, and a frequency of 1 kHz.
It is preferable to perform the process at a repetition frequency of z and an output of 30 mW / cm ² .

The ultrasonic wave generator for irradiating the ultrasonic wave of the present invention is not particularly limited. For example, in the case of clinical application, it is described in US Pat. No. 4,530,360 to Duarte. It is possible to use a basic non-invasive treatment device or the like that can apply an ultrasonic pulse percutaneously from outside the body close to a diseased part. The ultrasonic wave generated from the device has a frequency of 1.3 to 2 MHz and a repetition period of 100 to 1,
It is a low-power ultrasonic wave of 000 Hz, a pulse width of 10 to 2000 μsec, and an output of 100 mW / cm ² or less. The irradiation time is preferably within 20 minutes per day. Also, an ultrasound body treatment system having a body applicator unit connected to a remote control unit according to the US Patent No. 5,003,965 to Talish et al. Can be used. The ultrasonic waves in this case are low-power ultrasonic waves having a frequency of 1.3 to ² MHz and an output of 1 to 50 mW / cm ² .

By applying the method of inhibiting osteoclast formation or bone resorption of the present invention, an ultrasonic wave is applied to an affected area where bone loss or bone destruction occurs due to osteoclast formation or enhanced bone resorption. For the treatment of bone-related diseases such as osteoporosis, bone loss or destruction of rheumatoid arthritis, bone loss immediately after artificial joint replacement, loosening after artificial joint replacement, bone destruction due to tumor metastasized to bone, etc. Clinical application becomes possible. Furthermore, the method of inhibiting osteoclast formation or bone resorption of the present invention has a low risk of side effects due to the administration of a drug or the like, which has been a problem in the past, and is expected to have clinical applications in the future.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to examples.

Examples 1-1 to 1-4 and Comparative Example 1
1-1-1-4] Mouse bone marrow-derived osteoclast precursor cells (hereinafter referred to as “MDBM”) are obtained by collecting the bone marrow of the femur and tibia of a mouse and then separating only monocyte components by a conventional method.
× 106 cells / 10cm-dish seeded and only adherent cells 100 n
It was obtained by culturing for 3 days in the presence of g / ml M-CSF. The medium used was an alpha MEM medium containing 100 ng / ml M-CSF and supplemented with 10% fetal bovine serum. MDBM 1 × 105 / well
At a density of 2 ml in 6-wells. Here, soluble RANKL (hereinafter, referred to as "sRANKL") was added to a concentration of 50 ng / ml. sRANKL used was obtained from Pepro-Tech. The experiment set up the following groups.

Comparative Example 1-1; Control group (control)
72-hour culture group of Example 1-1; 72-hour culture group of ultrasonic irradiation group Ultrasonic waves were irradiated from the bottom of the wells for 20 minutes a day for 2 consecutive days. Comparative Example 1-2; 96-hour culture group of control group (control) Example 1-2; 96-hour culture group of ultrasonic irradiation group Ultrasonic wave was irradiated from the bottom of the well for 20 minutes a day for 3 consecutive days. Comparative Example 1-3; 108-hour culture group of control group (control) Example 1-3; 108-hour culture group of ultrasonic irradiation group Ultrasonic waves were irradiated from the bottom of the wells for 20 minutes a day for 4 consecutive days. Comparative Example 1-4; 144-hour culture group of control group (control) Example 1-4; 144-hour culture group of ultrasonic irradiation group Ultrasonic waves were irradiated from the bottom of the well for 20 minutes a day for 5 consecutive days.

Regarding the addition of sRANKL, in each of Comparative Examples and Examples, a solution dissolved in sterilized distilled water to a concentration of 10 μg / ml was added so as to be 50 ng / ml in 200-fold dilution. On the third day, both the comparative example and the example were replaced with fresh 10% FCS-αMEM containing 50 ng / ml of sRANKL.

Regarding the ultrasonic irradiation, Comparative Examples 1-1 to 1-1
In 1-4, ultrasonic waves were not irradiated, and Examples 1-1 to 1-3 were not applied.
Now, low power ultrasonic pulse for 20 minutes a day, Exogen
In Example 1-1, two consecutive days were used from the bottom of the well using a modified ultrasonic output unit for cell irradiation.
Irradiation was performed for 3 consecutive days in Example 1-2, for 4 consecutive days in Example 1-3, and for 5 consecutive days in Example 1-4. Here, the output characteristics of the ultrasonic wave are: burst width 200 μsec, ultrasonic frequency
The test was performed at a repetition frequency of 1.5 MHz and 1 kHz and an output of 30 mW / cm ² .

In Comparative Example 1-1 and Example 1-1, 72 hours after the start of the culture, in Comparative Example 1-2 and Example 1-2, 96 hours after the start of the culture, Comparative Example 1-3 and Example 1-3 108 hours after the start of culture, Comparative Example 1-4 and Example 1-4
In 144 hours after the start of the culture, the medium was removed, the cells were fixed with phosphate buffered formalin, and tartrate-resistant acid phosphatase (TRAP), a marker for osteoclasts, was stained according to a conventional method. Having more than one cell nucleus
The number of TRAP-positive multinucleated cells was determined as osteoclasts.

The results are shown in FIG. As is clear from FIG. 1, the number of osteoclasts in Examples 1-2 to 1-4 is smaller than that in Comparative Examples 1-2 to 1-4.

[Examples 2-1 to 2-2 and Comparative Example 2-
1-2-2] MDBM is obtained by collecting bone marrow of femur and tibia of a mouse, and then separating only monocyte components by a conventional method.
06 cells / 10cm-dish seeded and only adherent cells 100 ng / m
It was obtained by culturing for 3 days in the presence of 1 M-CSF. The medium used was an alpha MEM medium containing 100 ng / ml M-CSF and supplemented with 10% fetal bovine serum. Mouse bone marrow-derived stromal cell line TSB-13-9 is SV-40-large-T-antigen transg
A cell line established from the bone marrow of enic mice. TSB-13-9
Was used by inoculating 2 × 10 5 cells / 10 cm-dish and culturing for 3 days. As a medium, an alpha MEM medium supplemented with 10% fetal bovine serum was used.

The MDBM has a density of 1 × 105 cells / well and a TSB-13-9
Was seeded at a density of 1 × 10 6 cells / well and simultaneously in a 6-well volume in an amount of 2 ml. To this, 1 × 10 ⁻⁸ M 1α, 25-dihydroxyvitamin D3 was added. The experiment set up the following groups.

Comparative Example 2-1: Control group (control)
96-hour culture group of Example 2-1; 96-hour culture group of ultrasonic irradiation group Ultrasonic wave was irradiated from the bottom of the well for 20 minutes a day for 3 consecutive days. Comparative Example 2-2; 120-hour culture group of control group (control) Example 2-2; 120-hour culture group of ultrasonic irradiation group Ultrasonic waves were irradiated from the bottom of the wells for 20 minutes a day for 4 consecutive days.

With respect to the addition of 1α, 25-dihydroxyvitamin D3, ethanol was added to ethanol in both Comparative Examples and Examples.
Dissolved to a concentration of × 10 ^-5 M, diluted 1000 times to 1 × 10 ^-8
M was added. On the third day, both the comparative example and the example were replaced with fresh 10% FCS-αMEM containing 1 × 10 ⁻⁸ M 1α, 25-dihydroxyvitamin D3.

With respect to the ultrasonic irradiation, Comparative Examples 1-1 to 1-1
In 1-2, ultrasonic waves were not irradiated, and Examples 1-1 to 1-2 were used.
Now, low power ultrasonic pulse for 20 minutes a day, Exogen
In Example 1-1, irradiation was performed continuously for 3 days from the bottom of the well, and in Example 1-2, irradiation was performed continuously for 4 days using a modified ultrasonic output unit for cell irradiation. here,
The output characteristics of the ultrasonic wave were measured at a burst width of 200 μsec, an ultrasonic frequency of 1.5 MHz, a repetition frequency of 1 kHz, and an output of 30 mW / cm ² .

In Comparative Example 1-1 and Example 1-1, the medium was removed 96 hours after the start of the culture, and in Comparative Example 1-2 and Example 1-2, the medium was removed 120 hours after the start of the culture. After fixation with formalin, tartrate-resistant acid phosphatase (TRAP)
That. ) Staining was performed, and the number of TRAP-positive multinucleated cells having 10 or more cell nuclei was determined as osteoclasts under an optical microscope. The results are shown in FIG.

As is clear from FIGS. 2 and 3, the number of osteoclasts in Examples 1-1 to 1-2 was determined in Comparative Examples 1-1 to 1-2.
It is smaller than 1-2.

[Example 3-1 and Comparative Example 3-1] MDBM prepared in the same manner as in Example 1 was seeded at a density of 2 × 10 ⁴ cells / well in a 24-well well in an amount of 1 ml. In the 24-wells, a hydroxyapatite-coated disc (Osteologic, Millenniun Bi.
ologix, Inc.), and cells were allowed to survive. sRANKL was added to 50 ng / ml.
sRANKL used was obtained from Pepro-Tech.

The experiments set up the following groups: Comparative Example 3-1; 120-hour culture group of control group (control) Example 3-1; 120-hour culture group of ultrasonic irradiation group Ultrasonic waves were irradiated from the bottom of the well for 20 minutes a day for 4 consecutive days.

Regarding the addition of sRANKL, in each of Comparative Examples and Examples, a solution dissolved in sterilized distilled water to a concentration of 10 μg / ml was added so as to be 50 ng / ml in 200-fold dilution. On the third day, both the comparative example and the example were replaced with fresh 10% FCS-αMEM containing 50 ng / ml of sRANKL.

Regarding the irradiation of ultrasonic waves, in Comparative Example 3-1, no ultrasonic waves were irradiated, and in Example 3-1 a low-output ultrasonic pulse was applied for 20 minutes a day, and an ultrasonic output unit of Exogen was used to apply a cell. In Example 3-1 the sample was irradiated for 4 consecutive days from the bottom of the well using a sample modified for irradiation. here,
The output characteristics of the ultrasonic wave were measured at a burst width of 200 μsec, an ultrasonic frequency of 1.5 MHz, a repetition frequency of 1 kHz, and an output of 30 mW / cm ² .

In Comparative Example 3-1 and Example 3-1, the medium was removed 120 hours after the start of the culture, and the hydroxyapatite-coated disc was taken out. After washing with distilled water, the dissolved state of hydroxyapatite by the osteoclasts was observed under an optical microscope. A part of the disk (50% or more of the entire area) was loaded into a computer by an image capturing device, and the dissolved area was measured using image analysis software. The dissolved area per total area was calculated and is shown in FIG. 3 as the area of bone resorption by osteoclasts.

As is apparent from FIG. 3, the area of bone resorption by osteoclasts in Example 3-1 is smaller than that in Comparative Example 3-1.

[Example 4-1 and Comparative Example 4-1] MDBM and mouse bone marrow-derived stromal cell line TSB-13-9 were prepared in the same manner as in Example 2. As a medium, an alpha MEM medium supplemented with 10% fetal bovine serum was used.

The MDBM has a density of 2 × 10 ⁴ cells / well and a TSB-13-9
Were seeded simultaneously into 24-wells at a density of 1 × 10 ⁵ cells / well. The medium volume was finally adjusted to 1 ml. In the 24-well, a hydroxyapatite-coated disk (Osteologic, Millenniun B
iologix, Inc.), and cells were allowed to survive. Where 1 is the final concentration of 1 × 10 ^-8 M
alpha, it was added 25-dihydroxyvitamin D _3.

In the experiment, the following groups were set. Comparative Example 4-1; 120-hour culture group of control group (control) Example 4-1; 120-hour culture group of ultrasonic irradiation group Ultrasonic waves were irradiated from the bottom of the well for 20 minutes a day for 4 consecutive days.

[0039] l [alpha], 25-regard to the addition of dihydroxyvitamin D _3, in both Comparative Examples and Examples, ethanol 1
Dissolved to a concentration of × 10 ^-5 M, diluted 1000 times to 1 × 10 ^-8
M was added. Both Comparative Examples and Examples 3 day 1 × 10 ^-8 M of l [alpha], including the 25-dihydroxyvitamin D ₃ was replaced with fresh 10% FCS-αMEM.

Regarding the irradiation of ultrasonic waves, in Comparative Example 4-1, no ultrasonic waves were irradiated, and in Example 4-1 a low-output ultrasonic pulse was applied for 20 minutes a day. In Example 4-1, irradiation was performed for 4 consecutive days from the bottom of the well using the one modified for irradiation. here,
The output characteristics of the ultrasonic wave were measured at a burst width of 200 μsec, an ultrasonic frequency of 1.5 MHz, a repetition frequency of 1 kHz, and an output of 30 mW / cm ² .

In Comparative Example 4-1 and Example 4-1, the medium was removed 120 hours after the start of the culture, and the hydroxyapatite-coated disc was taken out. After washing with distilled water, the dissolved state of hydroxyapatite by the osteoclasts was observed under an optical microscope. A part of the disk (50% or more of the entire area) was loaded into a computer by an image capturing device, and the dissolved area was measured using image analysis software. The dissolved area per total area was calculated and is shown in FIG. 4 as the area of bone resorption by osteoclasts.

As is clear from FIG. 4, the area of bone resorption by osteoclasts in Example 4-1 is smaller than that in Comparative Example 4-1.

[0043]

The present invention relates to a method for inhibiting osteoclast formation or bone resorption characterized by irradiating an ultrasonic wave. Such a physical / non-invasive ultrasonic wave is applied to an osteoclast. By irradiating the affected area with bone loss or destruction due to formation or bone resorption, there is no risk of side effects and bone-related diseases such as osteoporosis, bone loss or destruction in rheumatoid arthritis, artificial joints Clinical application for the treatment of bone loss immediately after the replacement, loosening after the artificial joint replacement, bone destruction due to a bone metastasis tumor, and the like becomes possible. Furthermore, the method of inhibiting osteoclast formation or bone resorption of the present invention has a low risk of side effects due to administration of a drug or the like, which has been a problem in the past, and is expected to have clinical applications in the future.

[Brief description of the drawings]

FIG. 1 shows the number of osteoclasts formed by sRANKL from mouse osteoclast precursor cells, and shows the time-dependent change in the number of osteoclasts.

FIG. 2 shows 96 cells formed by co-culture of mouse osteoclast precursor cells and mouse bone marrow stromal cell line.
It shows the number of osteoclasts after time.

FIG. 3 shows 12 cells formed by co-culture of mouse osteoclast precursor cells and mouse bone marrow stromal cell line.
It shows the number of osteoclasts at 0 hours.

FIG. 4 shows the area of bone resorption by osteoclasts formed by co-culture of mouse osteoclast precursor cells and a mouse bone marrow stromal cell line.

[Explanation of symbols]

* Indicates p <0.0 against the control group as a result of Student * s t-test.
Indicates that it is 5. *** is the result of Student * s t-test,
It represents that p <0.001 with respect to the control group.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Keiji Komoriya 4-3-2 Asahigaoka, Hino-shi, Tokyo Teijin, Ltd. Tokyo Research Center Co., Ltd. (72) Inventor Akira Kudo 1-15-20, Nishi 1-chome, Kunitachi-shi, Tokyo (72) Inventor Atsushi Takeshita 2129-3 Shimotsuruma, Yamato-shi, Kanagawa Pref. 2-904 Tsukimino, Lions Mansion 4B065 AA90X AA91X BB19 BB34 BC50 CA46 4C084 AA02 BA44 DA12 DA13 DA15 DA16 DA18 DA19 DA25 DB32 DB60 ZA512 ZA962 ZC232 4C086 AA01 AA02 DA01 DA15 MA01 MA04 NA05 ZA51 ZA96 ZC23

Claims (6)

[Claims] 1. A method for inhibiting osteoclast formation or inhibiting bone resorption, which comprises irradiating an ultrasonic wave in a culture system containing a culture solution containing osteoclast precursor cells and inducing factors. 2. The osteoclast according to claim 1, wherein the inducers are at least one selected from macrophage colony stimulating factor, osteoclast-forming factor, tumor necrosis factor, interleukin-4, and vascular endothelial cell growth factor. Cell formation suppressing method or bone resorption suppressing method. 3. A method for inhibiting osteoclast formation or inhibiting bone resorption, which comprises irradiating an ultrasonic wave in a co-culture system containing a culture solution containing osteoclast precursor cells. 4. The method according to claim 3, wherein the cells contained in the co-culture system are at least one selected from osteoblasts, stromal cells, fibroblasts, T cells, and B cells. Bone resorption control method. 5. The method for inhibiting osteoclast formation or bone resorption according to claim 3, wherein the culture medium of the co-culture system contains an inducing factor. 6. The inducers include macrophage colony stimulating factor, osteoclast-forming factor, tumor necrosis factor, interleukin-1, interleukin-3, interleukin-4, interleukin-6, and interleukin-.
11, interleukin-15, interleukin-1
7. Prostaglandins, parathyroid hormone, parathyroid hormone-related peptide, vascular endothelial cell growth factor, granulocyte / macrophage colony stimulating factor, active vitamin
The method for inhibiting osteoclast formation or the method for inhibiting bone resorption according to claim 5, wherein the method is at least one selected from Class D.