WO2015108088A1 - Phototherapeutic device for metabolic bone diseases - Google Patents

Abstract

The present invention addresses the problem of providing a phototherapeutic device for metabolic bone diseases, which enables the remedy of symptoms of metabolic bone diseases. The present invention relates to a phototherapeutic device (1) for metabolic bone diseases, which is equipped with at least light source units, wherein the light source units are respectively equipped with light sources (7, 8) each capable of outputting healing light of which the main wavelength bandwidth is near-infrared light, and wherein the light source units can emit healing light output from the light sources (7, 8) onto an affected site (5).

Description

Phototherapy device for metabolic bone disease

The present invention relates to a phototherapy device for metabolic bone disease.

Metabolic bone disease is a disease in which the balance of bone metabolism by osteoblasts (Osteoblast) that form bone in bone tissue and osteoclasts (Osteoblast) that destroy bone is disrupted. Examples of this metabolic bone disease include osteoporosis, osteomalacia, and marble disease. In addition, patients with rheumatoid arthritis often have metabolic bone diseases such as osteoporosis.

Conventionally, for example, a phototherapy device for rheumatoid arthritis has been proposed (for example, Patent Document 1). This phototherapy device dilates blood vessels by irradiating the affected area such as the finger joints of rheumatoid arthritis patients with treatment light with a near-infrared light that is highly penetrating into the living body, and functions of peripheral nerves. By improving the above, it is possible to achieve pain relief and anti-inflammatory effect.

Japanese Unexamined Patent Publication No. 2009-207605

The above-mentioned conventional technique realizes a pain relief and anti-inflammatory effect for a certain period of time by irradiation with therapeutic light to the affected area, and does not improve the symptoms of the disease itself. There is a need for a technique to improve this.

Therefore, an object of the present invention is to provide a phototherapy device for metabolic bone disease that can improve the symptoms of metabolic bone disease.

The present invention is as follows.
1. A phototherapy device for metabolic bone disease, comprising: a light source that outputs treatment light having near-infrared light as a main wavelength band; and at least a light source unit that irradiates the affected part with the treatment light output from the light source.
2. The phototherapy device for metabolic bone disease according to 1, wherein the light source unit includes a plurality of light sources, and the treatment light output from each of the plurality of light sources is irradiated onto the affected area.
3. 3. The phototherapy device for metabolic bone disease according to 1 or 2, wherein the light source unit irradiates the affected part with treatment light including blue light.
4). 4. The phototherapeutic device for metabolic bone disease according to any one of 1 to 3, wherein the near infrared light is light having a wavelength of 700 to 900 nm.
5. 5. The metabolic bone disease according to any one of the above 1 to 4, wherein the metabolic bone disease is an articular bone or cartilage degenerative joint disease associated with any one selected from osteoporosis, Paget's disease, rheumatoid arthritis, and osteoarthritis Phototherapy device for metabolic bone disease.

According to the phototherapy device for metabolic bone disease of the present invention, the symptoms of metabolic bone disease itself can be improved.

FIG. 1 is an auxiliary diagram for explaining a flow of operations of a light irradiation experiment using cultured cells in the first embodiment. FIGS. 2A to 2C are diagrams showing measurement results of IL-6 production of synovial fibroblasts derived from knee osteoarthritis patients in Experiment 1-1 of Embodiment 1. FIG. . FIGS. 3 (a) to 3 (c) are diagrams showing measurement results of the amount of MMP-3 produced by synovial fibroblasts derived from patients with knee osteoarthritis in Experiment 1-2 of Embodiment 1. FIG. . FIG. 4 is a diagram showing the measurement results of the amount of MMP-13 produced by synovial fibroblasts derived from patients with knee osteoarthritis in Experiment 1-2 of Embodiment 1. 5 (a) and 5 (b) are diagrams showing measurement results of ALP activity of normal osteoblasts in Experiment 2-1 of Embodiment 1. FIG. 6 (a) and 6 (b) are diagrams showing measurement results of the amount of osteocalcin produced by normal osteoblasts in Experiment 2-2 of Embodiment 1. FIG. FIGS. 7A and 7B are diagrams showing the results of measuring the number of osteoclasts in Experiment 3-1-1 of Embodiment 1. FIG. FIGS. 8A and 8B are diagrams showing measurement results of the degree of dissolution of dentin sections by osteoclasts in Experiment 3-1-2 of Embodiment 1. FIG. FIGS. 9A to 9C are diagrams showing an example of an image of a dentin section dissolved by osteoclasts in Experiment 3-1-2 of Embodiment 1. FIG. 10 (a) and 10 (b) are diagrams showing the results of measuring the number of osteoclasts in Experiment 3-2-1 of Embodiment 1. FIG. FIG. 11 is a diagram showing measurement results of the degree of dissolution of dentin slices by osteoclasts in Experiment 3-2-2 of Embodiment 1. FIG. 12 is a diagram showing measurement results of cartilage matrix proteoglycan production of chondrocytes derived from patients with knee osteoarthritis in Experiment 3-3 of Embodiment 1. FIGS. 13A and 13B are images showing the healthy rat and the left hind limb of the CIA model in the second embodiment. FIG. 14 is a diagram illustrating a measurement result of the thickness of the hind limb portion of the CIA model in Experiment 5-1-1 of the second embodiment. FIG. 15 is a diagram illustrating a measurement result of the behavior amount of the CIA model in the experiment 5-1-2 of the second embodiment. FIGS. 16A to 16C are diagrams showing an example of measurement results of the trajectory of the rat's behavior in the experiment 5-1-2 of the second embodiment. FIGS. 17A to 17C are images of HE-stained pathological specimens of the longitudinal cross section of the right hind knee joint of the CIA model according to Experiment 5-2 of the second embodiment. FIGS. 18A to 18C are X-ray image images of the short-axis cross-section of the left limb femur of the CIA model in Experiment 6-1 of Embodiment 2. FIG. FIG. 19 is a diagram illustrating measurement results of luminance values of the X-ray image of the short-axis cross section of the left limb femur of the CIA model in Experiment 6-1 of the second embodiment. FIGS. 20A to 20C are images of pathological staining of the epiphyseal site of the femur of the right hind limb of the CIA model in Experiment 6-2 of the second embodiment. 21A to 21C are images of pathological staining of the epiphyseal site of the femur of the right hind limb of the CIA model in Experiment 6-3 of the second embodiment. FIG. 22 is a perspective view of the phototherapy device for metabolic bone disease used in Experiment 7 of Embodiment 3. FIG. 23 is a cross-sectional view of a phototherapy device for metabolic bone disease used in Experiment 7 of Embodiment 3. FIG. 24 is a diagram illustrating a usage state of the phototherapy device for metabolic bone disease used in Experiment 7 of the third embodiment. FIG. 25 is an MRI image of the finger portion of the first patient in Experiment 7 of the third embodiment. FIG. 26 is an MRI image of the finger part of the second patient in Experiment 7 of the third embodiment. FIG. 27 is an MRI image of the finger part of the third patient in Experiment 7 of the third embodiment.

The phototherapy device for metabolic bone disease of the present invention includes a light source that outputs treatment light having a near-infrared light as a main wavelength band, and at least a light source unit that irradiates the affected part with the treatment light output from the light source. Have.

The “treatment of metabolic bone disease” as used in the present invention means improvement of the state of metabolic bone disease itself. That is, to improve the state of metabolic bone disease itself means to suppress the progression of bone density decrease due to metabolic bone disease, to stop the progression of bone density decrease, or to increase bone density. Say.

Examples of metabolic bone diseases include osteoporosis, Paget's disease of bone, rheumatoid arthritis, or joint disease associated with cartilage degeneration associated with osteoarthritis.

The reason why the phototherapy device for metabolic bone disease of the present invention is effective for osteoporosis and Paget's disease of bone will be described below.

Bisphosphonates (eg, etidronate disodium or risedronate sodium hydrate) have been used as a treatment for osteoporosis or Paget's disease of osteoporosis. It is thought to act and suppress bone resorption.

First, bisphosphonates bind strongly to hydroxyapatite and deposit on the bone surface. Osteoclasts release protons (H ⁺ ) to the bone surface via the vacuolar proton ATPase to form a bone resorption fossa. However, since the bone resorption flotation is an acidic environment, the bisphosphonate is released from the bone. And is taken up by osteoclasts. Incorporated bisphosphonates are thought to induce apoptosis by inhibiting farnesyl pyrophosphate synthase in osteoclasts and suppress bone resorption.

In the examples described later, it was confirmed that irradiation to light suppresses differentiation into mature osteoclasts and activation of osteoclasts by an experiment using osteoclasts. Moreover, the result of suppressing the bone density decrease of a rat by irradiating light by animal experiment was shown.

These results indicate that, like bisphosphonates, light acts on osteoclasts and has the effect of suppressing bone resorption. Therefore, the phototherapy device for metabolic bone disease of the present invention is considered to be effective for the treatment of osteoporosis and Paget's disease of bone.

The reason why the phototherapy device for metabolic bone disease of the present invention is effective for articular bone and cartilage degeneration joint diseases associated with rheumatoid arthritis and osteoarthritis will be described below.

Rheumatoid arthritis is a chronic inflammatory disease whose synovial membrane is the main component of inflammation, and its destruction progresses not only to articular cartilage but also to bone, whereas osteoarthritis associated with aging or joint overload is a tissue However, both diseases are characterized by joint destruction with arthritis.

It has been found that matrix metalloproteinases that degrade cartilage matrix such as MMP-3 or MMP-13 are produced from synovial cells or chondrocytes denatured by inflammation, and that joint cartilage is degraded and bone destruction proceeds.

In the examples described below, it was confirmed that the production of MMP-3 or MMP-13 was suppressed by irradiating light from an experiment using synovial cells, and the cartilage destruction was also suppressed from observation of pathological specimens in animal experiments. It is shown. Furthermore, in the examples described later, it has been shown from experiments using chondrocytes that the production of cartilage matrix proteoglycans from chondrocytes is promoted by irradiation with blue light (470 nm). Therefore, it is considered that the phototherapy device for metabolic bone disease of the present invention is effective for the treatment of articular bone and cartilage degeneration joint diseases associated with rheumatoid arthritis and osteoarthritis.

The reason why near-infrared light is used as the main wavelength region as the treatment light in the present invention is that the near-infrared light is useful for improving the balance of bone metabolism as shown in the experimental results of Embodiments 1 to 3 described later. In addition, since it is difficult to be absorbed by hemoglobin in water or blood, treatment light can be supplied to the deep tissue of the affected area.

Hereinafter, the conditions of treatment light having near-infrared light as a main wavelength band used for the treatment of metabolic diseases will be described. In addition, the following conditions can be appropriately selected from the range in consideration of the patient's sex, age, height, weight, disease symptoms, and the like.

In the present invention, near-infrared light refers to a wavelength of approximately 700 to 900 nm. In the present invention, it is preferable that the treatment light irradiation with near infrared light as a main wavelength band is percutaneous irradiation. In the present invention, the treatment light having near infrared light as the main wavelength band can be continuous irradiation or pulse irradiation.

The light source unit of the phototherapeutic device for metabolic bone disease of the present invention outputs therapeutic light having a near-infrared light as a main wavelength band from the light source from a plurality of light sources, and irradiates the affected area to the affected area with a therapeutic effect From the viewpoint of The surface irradiation referred to in the present invention means that the region irradiated with the treatment light is not a point but a surface. It is preferable that the treatment light is irradiated substantially uniformly over the entire affected area, and local irradiation is not recognized.

Note that the treatment light of the present invention is not limited to near-infrared light, and examples thereof include treatment light having a wavelength band of approximately 300 to 1000 nm. Of the treatment light in the above wavelength band, for example, blue light indicates a wavelength of approximately 430 to 500 nm, and red light indicates a wavelength of approximately 600 to 750 nm.

Further, the light source in the phototherapy device for metabolic bone disease of the present invention can be a known light source that satisfies the above-mentioned conditions, and the light source can be incorporated in a known irradiation apparatus. Examples of such a known irradiation apparatus include INNOVIVE MED MULTI WAVE LIGHT THERAPY SYSTEM (manufactured by innovative MED INC.).

Hereinafter, a phototherapy device for metabolic bone disease according to one aspect of an embodiment of the present invention will be described with reference to the drawings.
In the following embodiments, rheumatoid arthritis and osteoarthritis complicated with metabolic bone disease are described as examples. However, the present invention is not limited to this, and is generally applicable to metabolic bone diseases. Can be used.

(Embodiment 1)
In the first embodiment, it is shown by an experiment using cultured cells that treatment light having near-infrared light as a main wavelength band is useful for improving symptoms of metabolic bone disease itself.

≪Experiment 1≫
In the experiment 1, light in synovial fibroblasts (synovial fibroblasts) in which inflammation was artificially induced by adding interleukin-1β (hereinafter abbreviated as “IL-1β”). The impact of.

Specifically, in Experiment 1, synovial fibroblasts interleukin-6 (Interleukin-6) with or without irradiation of synovial fibroblasts in which inflammation was induced by IL-1β were irradiated. , Hereinafter abbreviated as “IL-6”), matrix metaproteinase-3 (Matrix Metalloproteinase-3, hereinafter abbreviated as “MMP-3”) and matrix metaproteinase-13 (Matrix Metalloprotease-13, hereinafter) , Abbreviated as “MMP-13”), and the influence of light was evaluated. IL-6, MMP-3 and MMP-13 are not markers for evaluating metabolic bone disease, but markers for evaluating the symptoms of rheumatoid arthritis itself.

The reason why IL-6 was used as the evaluation marker in Experiment 1 is as follows. IL-6 is one of the inflammatory markers produced when IL-1β causes inflammation in synovial fibroblasts, and is generally used for clinical evaluation of inflammation in rheumatoid arthritis.

Therefore, whether light has an anti-inflammatory effect of rheumatoid arthritis should be evaluated by comparing IL-6 production of synovial fibroblasts with and without light irradiation. Can do. Therefore, IL-6 was used as an evaluation marker in Experiment 1. This experiment was conducted to confirm the inflammation-inhibiting effect of light. Hereinafter, this experiment is referred to as “Experiment 1-1”.

The reason why MMP-3 and MMP-13 were used as the evaluation markers in Experiment 1 is as follows. MMP-3 and MMP-13 are enzymes produced with inflammation of synovial fibroblasts and have a function of lysing cartilage matrix.

It is known that the amount of MMP-3 and MMP-13 produced is increased in synovial fibroblasts in the initial or intermediate symptoms of rheumatoid arthritis. That is, an increase in the production amount of MMP-3 and MMP-13 from synovial fibroblasts indicates that cartilage lysis is in progress, resulting in possible damage to bone It turns out that it is.

Therefore, whether or not light has an effect of inhibiting cartilage dissolution at the initial stage of damage to bone depends on whether the amount of synovial fibroblasts produced MMP-3 and MM-13 is or is not irradiated with light. It can be evaluated by comparing Therefore, MMP-3 and MM-13 were used as evaluation markers in Experiment 1. Hereinafter, this experiment is referred to as “Experiment 1-2”.

Synovial fibroblasts used in Experiment 1 were patients with osteoarthritis of the knee (81 years old, female, stage 4 (stage classification based on bone destruction lesions as seen by X-ray examination by Steinblocker: stage 1 to 4) Collected from class 3 [classification of functional disorder based on daily activities by Steinblocker: classes 1 to 4)].

Further, the wavelength (nm) of light applied to the synovial fibroblasts in Experiment 1, the power density (mW / cm ² ) of the light applied to the synovial fibroblasts, and the irradiation of light applied to the synovial fibroblasts. The time (seconds) was measured under the 12 conditions shown in [Table 1] below.

In addition, the results of the experiment conducted under the above 12 conditions were synovial fibroblasts in which inflammation was induced by IL-1β, and the case where light irradiation was not performed was negative (hereinafter referred to as “negative control”). Based on a control that was synovial fibroblasts in which inflammation was not induced by IL-1β and was not irradiated with light (hereinafter referred to as “positive control”). Evaluation was performed.

<Procedure of Experiment 1>
Experiment 1 (Experiment 1-1 and Experiment 1-2) was performed according to the following procedure.

(1) The basic medium made by Sigma (Nutrient Mixture F-12 Ham, with L-glutamine and sodium bicarbonate, Sigma), and the fetal bovine serum (Vealbum made by EQUITEC BIO, INC) so that the final concentration is 10%. In the following, abbreviated as “FBS”) and Penicillin-Sterptomycine (5000 U / ml) manufactured by GIBCO were added so that the final concentration was 1% to prepare a first preparation medium.

(2) Synovial fibroblasts collected from patients were passaged 3 times at 37 ° C. in the first conditioned medium in a CO ₂ incubator.

(3) A second conditioned medium is prepared by adding IL-1β manufactured by R & D SYSTEMS so that the final concentration is 0.01 ng / ml in the first conditioned medium, and synovial fibroblasts are cultured. One conditioned medium was replaced with a second conditioned medium.

(4) As shown in FIG. 1, the transparent multiwell plate replaced with the second preparation medium was placed on the LED irradiation apparatus in the CO ₂ incubator. The prepared transparent multi-well plates were irradiated with light under each light irradiation condition.

(5) Each multiwell plate was stored in a storage CO ₂ incubator immediately after light irradiation.

(6) In each multi-well plate stored in a CO ₂ incubator, the supernatant of the culture solution in each well is collected after 2 hours, 6 hours, 12 hours, 24 hours and 48 hours after light irradiation. did.

(7) The concentrations of IL-6, MMP-3, and MMP-13 contained in the collected culture supernatant were measured by ELISA. The IL-6 concentration ELISA was performed using an R & D kit (IL-6, Human, ELISA kit Quantikine, model number S6050), and the MMP-3 concentration ELISA was performed using an R & D kit (MMP-3, Total A human ELISA kit Quantikine, SMP300) was used, and an RMP kit (MMP-13, Pro-human ELISA kit Quantikine, SMP1300) was used as the MMP-13 concentration ELISA.

<Evaluation result of Experiment 1>
1. Evaluation Results of Experiment 1 FIGS. 2 (a) to (c) show measurement results of IL-6 production by synovial fibroblasts derived from patients with knee osteoarthritis. FIG. 2 (a) shows changes over time in IL-6 production of synovial fibroblasts irradiated with near infrared light (840 nm). FIG. 2 (b) shows the change over time in IL-6 production of synovial fibroblasts irradiated with red light (660 nm). FIG. 2 (c) shows the time course of IL-6 production of synovial fibroblasts irradiated with blue light (470 nm).

From the results of FIGS. 2 (a) to 2 (c), when synovial fibroblasts are irradiated with light having a power density of 1 mW / cm ² for 50 seconds after 24 hours have passed since the synovial fibroblasts are irradiated with light. [Black triangles in FIGS. 2 (a) to (c)], when irradiated with light having a power density of 1 mW / cm ² for 500 seconds [Δ in FIGS. 2 (a) to (c)], power density of 10 mW / cm ² for 50 seconds [black squares in FIGS. 2 (a) to 2 (c)] and for light having a power density of 10 mW / cm ² for 500 seconds [in FIGS. 2 (a) to (c) The production amount of IL-6 in □ of □ is slight in the case of the negative control in any of near-infrared light (840 nm), red light (660 nm) and blue light (470 nm) [FIG. Compared with ○] in a) to (c), I of synovial fibroblasts It can be understood that the production amount is less than that of L-6.

That is, the results of FIGS. 2 (a) to 2 (c) show that IL-6 production from synovial fibroblasts is induced by light irradiation, regardless of whether it is near infrared light, blue light, or red light. It can be said that it is suppressing. Therefore, from the results of Experiment 1-1, it was confirmed that the irradiation of light to the synovial fibroblasts had an inflammation suppressing effect.

2. Evaluation Results of Experiment 1-2 FIGS. 3 (a) to 3 (c) show the measurement results of the production amount of MMP-3 by synovial fibroblasts derived from knee osteoarthritis patients. FIG. 3 (a) shows changes over time in the amount of MMP-3 produced by synovial fibroblasts irradiated with near-infrared light (840 nm). FIG. 3 (b) shows the change over time in the amount of MMP-3 produced by synovial fibroblasts irradiated with red light (660 nm). FIG. 3 (c) shows the change over time in the amount of MMP-3 produced by synovial fibroblasts irradiated with blue light (470 nm).

From the results of FIGS. 3 (a) to 3 (c), when synovial fibroblasts are irradiated with light having a power density of 1 mW / cm ² for 50 seconds after 12 hours have passed since the synovial fibroblasts are irradiated with light. [Black triangles in FIGS. 3 (a) to 3 (c)], irradiation with light having a power density of 1 mW / cm ² for 500 seconds [Δ in FIGS. 3 (a) to (c)], power density of 10 mW / cm ² for 50 seconds [black squares in FIGS. 3 (a) to 3 (c)] and for light having a power density of 10 mW / cm ² for 500 seconds [in FIGS. 3 (a) to (c) The production amount of MMP-3 in [□] of the negative control in any of the case of near infrared light (840 nm), red light (660 nm) and blue light (470 nm) [FIG. 3 (a) to ( c) Production of MMP-3 in synovial fibroblasts compared to [] It can be understood that it is less than the raw amount.

That is, the results in FIGS. 3 (a) to 3 (c) show that MMP-3 production from synovial fibroblasts is suppressed by light irradiation regardless of near-infrared light, blue light, and red light. It can be said that it suggests that.

On the other hand, FIG. 4 shows the measurement results of the amount of MMP-13 produced 48 hours after irradiation of synovial fibroblasts derived from knee osteoarthritis patients.

IL-1β (−) shown in FIG. 4 indicates a case where inflammation is not induced in IL-1β by synovial fibroblasts, and IL-1β (+) indicates that synovial fibroblasts are induced by IL-1β. It shows the case where inflammation is induced. The LED (−) shown in FIG. 4 indicates a case where light is not irradiated to synovial fibroblasts. Therefore, IL-1β (−) and LED (−) in FIG. 4 show the result of positive control, and IL-1β (+) and LED (−) show the result of negative control.

From the results of FIG. 4, the amount of MMP-13 produced by synovial fibroblasts is negative regardless of whether the light is irradiated with near-infrared light (840 nm), red light (660 nm), or blue light (470 nm). It can be understood that there are fewer than controls.

That is, the results in FIG. 4 suggest that MMP-13 production from synovial fibroblasts is suppressed by light irradiation regardless of near-infrared light, blue light, or red light. I can say that.

From Experiment 1-2, it is possible to obtain the result that the production of MMP-3 and MMP-13 is suppressed from synovial fibroblasts by light irradiation, and the light has an effect of suppressing cartilage matrix melting. I was able to find a new possibility.

≪Experiment 2≫
In Experiment 1, it was confirmed that irradiation of synovial fibroblasts with light had an inflammation-inhibiting effect. In addition, the possibility that irradiation of synovial fibroblasts with light has an inhibitory effect on the production of MMP-3 and MMP-13 (that is, that irradiation of light has an inhibitory effect on cartilage matrix melting) is newly seen. It was issued.

Next, the effect of light on bone metabolism was examined. Experiment 2 evaluates the influence of light on osteoblasts related to bone formation in bone metabolism. Specifically, in Experiment 2, alkaline phosphatase (Alkaline Phosphatase, hereinafter abbreviated as “ALP”) produced from normal osteoblasts when or not normal osteoblasts were irradiated with light. The activity and the amount of osteocalcin produced were compared, and the effect of light on osteoblasts was evaluated.

Note that ALP and osteocalcin are markers that indicate the degree of osteoblast activity, and in particular, osteocalcin is known to be a marker that directly indicates bone formation. Therefore, in Experiment 2, ALP and osteocalcin were used as evaluation markers.

In addition, the irradiation conditions of the osteoblasts in Experiment 2 were the same as those in [Table 1] in Experiment 1. In addition, the results of the 12 experiments performed in Experiment 2 were evaluated using as a control the case where normal osteoblasts were not irradiated with light.

Hereinafter, the evaluation of ALP activity of normal osteoblasts will be described as “Experiment 2-1,” and the evaluation of osteocalcin production will be described as “Experiment 2-2.”

<Procedure of Experiment 2>
Experiment 2 was performed according to the following procedure.

1. Experimental procedure of Experiment 2-1 (1) In LONZA osteoblast basic medium OBM, LONZA ascorbic acid to a final concentration of 0.1%, LONZA FBS to a final concentration of 10%, and A third preparation medium supplemented with LONZA GA-1000 was prepared so that the final concentration was 0.1%.

(2) Human-derived osteoblasts (Cat #: CLCC-2538) manufactured by Lonza were subcultured twice at 37 ° C. in a third preparation medium in a CO ₂ incubator.

(3) A fourth conditioned medium prepared by adding LONZA hydrocortisone hemi-succinate to a final concentration of 200 nM and LONZA β-glycerophosphoric acid to a final concentration of 10 mM is prepared in a third conditioned medium. The third preparation medium in which the derived osteoblasts were cultured was replaced with a fourth preparation medium.

(4) The transparent multiwell plate exchanged with the fourth preparation medium was placed on the LED irradiation apparatus in the CO ₂ incubator. And it prepared and irradiated light on each light irradiation condition with respect to each transparent multiwell plate.

(5) Each multiwell plate was stored in a storage CO ₂ incubator immediately after light irradiation.

(6) In each multiwell plate stored in a CO ₂ incubator, the supernatant of the culture solution in each well was collected after 0 days, 9 days, 12 days, and 18 days after light irradiation.

(7) Evaluation of ALP activity contained in the collected culture supernatant was performed by staining osteoblasts using an ALP staining kit (Cat # AK20) manufactured by Primary Cell. The concentration of osteocalcin contained in the collected culture supernatant was measured by ELISA. The osteocalcin concentration was measured using an ELISA kit manufactured by Takara Bio Inc. (Human Gla-Osteocalcin High Sensitive EIA kit, Cat #: MK128).

<Evaluation results of Experiment 2>
1. Evaluation Results of Experiment 2-1 FIGS. 5 (a) and 5 (b) show measurement results of ALP activity produced from normal osteoblasts. FIG. 5 (a) shows the time course of ALP activity of normal osteoblasts after irradiating normal osteoblasts with light of each wavelength at a power density of 1 mW / cm ^{2 to} 10 mW / cm ² for 50 seconds. FIG. 5 (b) shows the change over time in the ALP activity of normal osteoblasts after irradiating normal osteoblasts with light of each wavelength at 1 mW / cm ^{2 to} 10 mW / cm ² for 500 seconds.

Note that “Area Fraction” on the vertical axis in FIGS. 5A and 5B is a ratio of the area occupied by osteoblasts exhibiting ALP activity within a certain visual field range. Since the stained cells can be evaluated as osteoblasts showing osteogenesis by staining the ALP produced simultaneously with the growth of the osteoblasts, the proportion of the stained osteoblasts within a certain visual field range (Area Fraction) ) Can be numerically calculated as the ratio of the area occupied by osteoblasts showing ALP activity within a certain visual field range.

From the results of FIGS. 5 (a) and 5 (b), normal osteoblasts were irradiated with near infrared light (840 nm) with a power density of 1 mW / cm ² for 50 seconds or 500 seconds [FIGS. 5 (a) and (b). ) In the case of irradiation with near infrared light (840 nm) having a power density of 10 mW / cm ² for 50 seconds or 500 seconds [□ in FIGS. 5A and 5B] It can be understood that the ALP activity produced from normal osteoblasts has almost no difference compared to the control [× in FIGS. 5 (a) and (b)]. That is, this result is considered to suggest that irradiation of near-infrared light (840 nm) to normal osteoblasts does not affect ALP activity.

On the other hand, from the results shown in FIGS. 5A and 5B, normal osteoblasts were irradiated with blue light (470 nm) having a power density of 1 mW / cm ² for 50 seconds or 500 seconds [FIGS. 5A and 5B. ) And blue light (470 nm) having a power density of 10 mW / cm ² is irradiated for 50 seconds or 500 seconds [normal circles in FIGS. 5 (a) and 5 (b)]. Compared with the ALP activity produced from normal osteoblasts compared to the control [X in FIG. 5 (a) and (b)] after 12 days from the blue light irradiation to the blast cells. It can be understood that the activity was greatly reduced.

From the results of FIGS. 5 (a) and 5 (b), normal osteoblasts were irradiated with red light (660 nm) having a power density of 1 mW / cm ² for 50 seconds or 500 seconds [FIGS. 5 (a) and (b). ) In black triangles] and when irradiated with red light (660 nm) with a power density of 10 mW / cm ² for 50 seconds or 500 seconds [Δ in FIG. 5 (a) and (b)], as a result of blue light, In any case, compared to the ALP activity in comparison with the control [X in FIG. 5 (a) and (b)] after 12 days after irradiation of red light to normal osteoblasts. It can be understood that the activity was greatly reduced.

That is, this result is considered to suggest that irradiation of blue light (470 nm) and red light (660 nm) to normal osteoblasts suppresses ALP activity.

From the results of FIG. 5, it was not possible to find that irradiation of near-infrared light to osteoblasts has an effect on the ALP activity of osteoblasts. On the other hand, irradiation of blue light and red light to osteoblasts resulted in a decrease in ALP activity of osteoblasts. That is, the experimental results indicate that at least blue light and red light may adversely affect osteoblast activity (ie, bone formation).

2. Evaluation Results of Experiment 2-2 FIGS. 6 (a) and 6 (b) show measurement results of the production amount of osteocalcin produced from normal osteoblasts. FIG. 6 (a) shows changes over time in the amount of osteocalcin produced by normal osteoblasts after irradiating normal osteoblasts with light of each wavelength at a power density of 1 mW / cm ^{2 to} 10 mW / cm ² for 50 seconds. . FIG. 6 (b) shows changes over time in the amount of osteocalcin produced by normal osteoblasts after irradiating normal osteoblasts with light of each wavelength at a power density of 1 mW / cm ^{2 to} 10 mW / cm ² for 500 seconds. .

From the results shown in FIGS. 6A and 6B, when normal osteoblasts were irradiated with near-infrared light (840 nm) having a power density of 1 mW / cm ² for 50 seconds or 500 seconds [FIGS. 6A and 6B] ) In the case of irradiation with near infrared light (840 nm) having a power density of 10 mW / cm ² for 50 seconds or 500 seconds [in FIG. 6 (a) and □ in (b)] It can be understood that the amount of osteocalcin produced was higher than in the case of the control [X in FIGS. 6 (a) and (b)]. That is, this result suggests that irradiation of near-infrared light (840 nm) to osteoblasts may activate osteoblasts and promote bone formation.

On the other hand, from the results shown in FIGS. 6A and 6B, normal osteoblasts were irradiated with blue light (470 nm) having a power density of 1 mW / cm ² for 50 seconds or 500 seconds [FIGS. 6A and 6B]. ) And blue light (470 nm) having a power density of 10 mW / cm ² is irradiated for 50 seconds or 500 seconds [normal circles in FIGS. 6 (a) and 6 (b)]. After the elapse of 8 days after irradiating blast cells with blue light, the amount of osteocalcin produced from normal osteoblasts was greatly reduced as compared to the control [X in FIGS. 6 (a) and (b)]. I understand that.

Further, from the results of FIGS. 6A and 6B, when normal osteoblasts were irradiated with red light (660 nm) having a power density of 1 mW / cm ² for 50 seconds or 500 seconds [FIGS. 6A and 6B] ) In black triangle] and when irradiated with light having a power density of 10 mW / cm ² for 50 seconds or 500 seconds [Δ in FIGS. 6A and 6B], as in the case of blue light, The amount of osteocalcin produced from normal osteoblasts compared to the control [X in FIG. 6 (a) and (b)] after 8 days after irradiation of red light to normal osteoblasts It can be understood that it has greatly decreased.

That is, this result suggests that irradiation of normal osteoblasts with blue light (470 nm) and red light (660 nm) may reduce osteoblast activity and suppress bone formation. ing.

From the results shown in FIGS. 6 (a) and 6 (b), it has been found that irradiation of near-infrared light to osteoblasts may improve osteoblast activity and promote bone formation. On the other hand, it has been found that irradiation of blue light and red light to osteoblasts may reduce osteoblast activity and suppress bone formation.

≪Experiment 3≫
Experiment 3 is an evaluation of the effect of light on osteoclasts related to osteolysis in bone metabolism. Before describing Experiment 3, the mechanism of bone destruction in rheumatoid arthritis will be described.

First, when inflammation is caused by rheumatoid arthritis, synovial fibroblasts present RANKL (Receptor Activator of Nuclear Factor Kappa-B Ligand), which is a protein involved in osteoclast formation and the like, on the cell surface. Next, osteoclast precursor cells (Osteoclast Precursor Cell) bind to RANKL presented on the surface layer of synovial fibroblasts and start to differentiate into mature osteoclasts (Mature Osteoblast).

In normal times (when rheumatoid arthritis has not developed), this mature osteoclast mainly resorbs bone, while osteoblasts form bone to maintain bone balance and maintain a certain balance. Is called. However, in the case of rheumatoid arthritis, TNF-α (Tumor Necrosis Factor) produced by excessive inflammation acts on mature osteoclasts.

Mature mature osteoclasts migrate to an active form that accelerates bone resorption by the action of TNF-α. Rheumatoid arthritis patients have an increased number of activated maturation cells and promote bone resorption compared to healthy individuals. As a result, bones in rheumatoid arthritis patients are damaged due to the collapse of the balance between bone formation and bone resorption.

In Experiment 3, whether or not light suppresses the differentiation of osteoclast precursor cells (hereinafter referred to as “Experiment 3-1”), and the light suppresses the change of osteoclasts to the active form. The effect of light on osteoclasts was evaluated by performing two evaluations: evaluation of whether or not (hereinafter referred to as “Experiment 3-2”).

<About Experiment 3-1>
As an experiment of Experiment 3-1, a first experiment (hereinafter referred to as “Experiment 3-1-1”) and a second experiment (hereinafter referred to as “Experiment 3-1-2”) were performed.

1. About Experiment 3-1-1 As the experiment of Experiment 3-1-1, the amount of tartrate-resistant acid phosphatase (Tartrate-Resitant Acid Phosphatase, hereinafter abbreviated as “TRAP”) produced from osteoclasts was measured. The effect of light on osteoclasts was evaluated.

The reason for using TRAP for evaluation is that osteoclasts differentiated by differentiation of osteoclast precursor cells generate TRAP, and the presence of osteoclasts can be determined by the generation of TRAP, so that light is differentiated from osteoclast precursor cells. This is because it can be evaluated whether or not the above is suppressed.

Specifically, the osteoclast precursor cells combined with RANKL were irradiated with light, mature osteoclasts differentiated after the light irradiation were TRAP stained, and the number of osteoclasts stained in red was counted.

The light irradiation conditions for the osteoclast precursor cells in Experiment 3-1-1 were the same as in [Table 1] in Experiment 1. In addition, the results of 12 experiments conducted in Experiment 3-1-1 evaluated the effect of light on osteoclasts, using as a control the case where light was not irradiated to osteoclast precursor cells.

2. Experiment 3-1-2 Experiment 3-1-2 was performed to confirm the experimental results of Experiment 3-1-1. Experiment 3-1-2 evaluates whether the osteoclast differentiated in Experiment 3-1-1 is resorbing bone.

Specifically, osteoclast precursor cells combined with RANKL are cultured on dentin slices, and the osteoclast precursor cells are irradiated with light, so that the degree of lysis of the differentiated osteoclasts in the dentin slices is increased. evaluated. When osteoclasts dissolve the dentin slice, the surface of the dentin slice becomes darker than other parts, so an image of the surface of the dentin slice is acquired at each time, and the dentin is obtained from the brightness value of the image. The area of the dissolved part of the section was calculated.

Note that the conditions for light irradiation to the osteoclast precursor cells in Experiment 3-1-2 are the same as in [Table 1] in Experiment 1. The results of 12 experiments conducted in Experiment 3-1-2 were evaluated as a control when no light was irradiated to osteoclast precursor cells.

<Procedure of Experiment 3-1>
Experiment 3-1 was performed according to the following procedure.

1. Procedures of Experiment 3-1-1 (1) LONZA L-Glutamate to a final concentration of 2 mM in LONZA human osteoclast precursor basic medium OBPM, LONZA FBS to a final concentration of 10% A fifth preparation medium supplemented with LONZA GA-1000 was prepared so that the final concentration was 1%.

(2) The human-derived osteoclast precursor cells (2T-110) manufactured by Lonza were cultured at 37 ° C. in a fifth preparation medium in a CO ₂ incubator.

(3) Prepare a sixth conditioned medium in which LONZA RANKL is added to the fifth prepared medium so that the final concentration is 30 ng / ml, and LONZA M-CSF is added so that the final concentration is 10 ng / ml. The fifth preparation medium in which human-derived osteoclast precursor cells were cultured was replaced with a sixth preparation medium.

(4) The transparent multiwell plate exchanged with the sixth preparation medium was placed on the LED irradiation apparatus in the CO ₂ incubator. The prepared transparent multi-well plates were irradiated with light under each light irradiation condition.

(5) Each multiwell plate was stored in a storage CO ₂ incubator immediately after light irradiation.

(6) In each multiwell plate stored in a CO ₂ incubator, osteoclasts cultured in each well 2 days, 4 days, 6 days, 8 days, 10 days, 12 days and 14 days after light irradiation Staining was performed with a TRAP staining kit (AK04F) manufactured by Primary Cell, and the number of stained osteoclasts was counted.

2. Procedure for Experiment 3-1-2

(1) LONZA human osteoclast precursor cell basic medium OBPM, LONZA L-Glutamate so that the final concentration is 2 mM, LONZA FBS so that the final concentration is 10%, and so that the final concentration is 1% A fifth prepared medium supplemented with LONZA GA-1000 was prepared.

(2) The human-derived osteoclast precursor cells (2T-110) manufactured by Lonza were cultured at 37 ° C. in a fifth preparation medium in a CO ₂ incubator.

(3) Prepare a sixth conditioned medium in which LONZA RANKL is added to the fifth prepared medium so that the final concentration is 30 ng / ml, and LONZA M-CSF is added so that the final concentration is 10 ng / ml. Human-derived osteoclast precursor cells cultured in a multiwell plate equipped with a dentin slice were seeded and replaced with a sixth preparation medium.

(4) The transparent multiwell plate exchanged with the sixth preparation medium was placed on the LED irradiation apparatus in the CO ₂ incubator. The prepared transparent multi-well plates were irradiated with light under each light irradiation condition.

(5) Each multiwell plate was stored in a storage CO ₂ incubator immediately after light irradiation.

(6) In each multiwell plate stored in a CO ₂ incubator, the dentin slices of each well after 2 days, 4 days, 6 days, 8 days, 10 days, 12 days and 14 days after light irradiation were collected. The degree of dissolution of the dentin slices by osteoclasts was observed using a microscope.

<Evaluation results of Experiment 3-1>
1. Evaluation Results of Experiment 3-1-1 FIG. 7 shows the measurement results of the number of osteoclasts stained red by TRAP staining. 7 (a) is, TRAP stained aging number of osteoclasts after the light of each wavelength is irradiated at a power density of 1 mW / cm ² to 10 mW / cm ² 50 seconds osteoclast precursor cells by binding RANKL Is shown. FIG. 7 (b) shows the time-dependent change in the number of osteoclasts stained with TRAP after irradiation with light of each wavelength to the osteoclast precursor cells combined with RANKL at a power density of 1 mW / cm ^{2 to} 10 mW / cm ² for 500 seconds. Show.

In the results shown in FIGS. 7A and 7B, osteoclast precursor cells combined with RANKL are irradiated with near-infrared light (840 nm) having a power density of 1 mW / cm ² for 50 seconds or 500 seconds [FIG. black squares in a) and (b)], when irradiated with near-infrared light (840 nm) having a power density of 10 mW / cm ² for 50 seconds or 500 seconds [□ in FIGS. 7 (a) and (b)], When red light (660 nm) with a power density of 1 mW / cm ² is irradiated for 50 seconds or 500 seconds [black triangles in FIGS. 7 (a) and (b)], red light (660 nm) with a power density of 10 mW / cm ² is applied. When irradiated for 50 seconds or 500 seconds [Δ in FIG. 7 (a) and (b)], when irradiated with blue light (470 nm) having a power density of 1 mW / cm ² for 50 seconds or 500 seconds [FIG. 7 (a) And ● in (b) And a power density 10 mW / cm ² of blue light (470 nm) the case of irradiation for 50 seconds or 500 seconds in either case of FIG. 7 (a) and 7 (b) ○ in, the case of the control [Figure 7 ( Compared with x] in a) and (b), the number of osteoclasts stained with TRAP was small.

That is, the result of FIG. 7 shows that osteoclast precursor cells are irradiated with near infrared light (840 nm), red light (660 nm), and blue light (470 nm) to differentiate osteoclast precursor cells into mature osteoclasts. It is suggested that this is suppressed.

2. Evaluation Results of Experiment 3-1-2 FIGS. 8A and 8B show the calculation results of the dissolution area of the dentin slice by osteoclasts. FIG. 8 (a) shows the change over time of the dissolution area of the ivory heterogeneous section by osteoclasts after irradiating the osteoclast precursor cells with light of each wavelength at a power density of 1 mW / cm ^{2 to} 10 mW / cm ² for 50 seconds. ing. FIG. 8 (b) shows the change over time of the dissolution area of the ivory heterogeneous section by osteoclasts after irradiating the osteoclast precursor cells with light of each wavelength at a power density of 1 mW / cm ^{2 to} 10 mW / cm ² for 500 seconds. Yes.

In the results shown in FIGS. 8A and 8B, osteoclast precursor cells combined with RANKL are irradiated with near-infrared light (840 nm) having a power density of 1 mW / cm ² for 50 seconds or 500 seconds [FIG. black squares in a) and (b)], when irradiated with near-infrared light (840 nm) having a power density of 10 mW / cm ² for 50 seconds or 500 seconds [□ in FIGS. 8A and 8B], When irradiated with red light (660 nm) having a power density of 1 mW / cm ² for 50 seconds or 500 seconds [black triangles in FIGS. 8A and 8B], red light having a power density of 10 mW / cm ² (660 nm) is emitted. When irradiated for 50 seconds or 500 seconds [Δ in FIGS. 8 (a) and (b)], when irradiated with blue light (470 nm) having a power density of 1 mW / cm ² for 50 seconds or 500 seconds [FIG. 8 (a) And ● in (b) And when blue light power density 10 mW / cm ² a (470 nm) Any when irradiated for 50 seconds or 500 seconds [○ in FIG. 8 (a) and (b)] is also the case of the control [Figure 8 ( Compared with x] in a) and (b), the dissolution area of the ivory heterogeneous section was small.

Results of FIGS. 8 (a) and 8 (b) showing that osteoclast progenitor cell differentiation was suppressed by light irradiation of Experiment 3-1-1 and that osteolysis by osteoclast cells was also suppressed by light irradiation. It was also possible to confirm.

As an example, FIGS. 9A to 9C show images of dentin slices obtained in Experiment 3-1-2. FIG. 9 (a) is an image on the 10th day from the start of culture on a dentin slice of osteoclast precursor cells in the case of control. FIG. 9 (b) is an image on the 14th day from the start of culture on the dentin slice of osteoclast precursor cells in the case of control. FIG. 9 (c) shows an image on the 14th day after the start of culture on the dentin slice of osteoclast precursor cells after irradiation of osteoclast precursor cells with near-infrared light (840 nm) having a power density of 10 mW / cm ² for 50 seconds. It is.

9A and 9B, which are the same sample, it can be understood that the dentin slice is dissolved on the 14th day compared to the 10th day from the start of the culture. Further, comparing FIG. 9B and FIG. 9C, which are samples having the same number of culture days, it can be understood that the dentin slice is dissolved in FIG. 9B.

<Experiment 3-2>
As the experiment 3-2, a first experiment (hereinafter referred to as “experiment 3-2-1”) and a second experiment (hereinafter referred to as “experiment 3-2-2”) were performed.

1. About Experiment 3-2-1 As an experiment of Experiment 3-2-1, light was irradiated to mature osteoclasts into which TNF-α had been introduced, and the experiment was carried out on mature osteoclasts after light irradiation. TRAP staining was performed in the same manner as in 1 and the number of stained osteoclasts was counted. The higher the number of stained osteoclasts, the more transition to the active form, so whether or not irradiation of light suppresses the transition of mature osteoclasts to the active form by irradiating light. Evaluated.

2. Experiment 3-2-2 Experiment 3-2-2 was performed to confirm the experimental results of Experiment 3-2-1. Experiment 3-2-2 is the same experiment as Experiment 3-1-2.

<Procedure of Experiment 3-2>
Experiment 3-2 was performed according to the following procedure.

1. Procedure for Experiment 3-2-1

(1) LONZA human osteoclast precursor cell basic medium OBPM, LONZA L-Glutamate so that the final concentration is 2 mM, LONZA FBS so that the final concentration is 10%, and so that the final concentration is 1% A fifth prepared medium supplemented with LONZA GA-1000 was prepared.

(2) Human-derived osteoclast precursor cells (2T-110) manufactured by LONZA were cultured at 37 ° C. in a fifth preparation medium in a CO ₂ incubator.

(3) Prepare a sixth conditioned medium in which LONZA RANKL is added to the fifth prepared medium so that the final concentration is 30 ng / ml, and LONZA M-CSF is added so that the final concentration is 10 ng / ml. The fifth preparation medium in which human-derived osteoclast precursor cells were cultured was replaced with the sixth preparation medium, and then cultured in the sixth preparation medium at 37 ° C. for 2 weeks in a CO ₂ incubator.

(4) A seventh prepared medium prepared by adding R & D SYSTEMS TNF-α to a final concentration of 10 ng / ml in the sixth prepared medium is prepared, and human-derived osteoclast precursor cells are cultured. The prepared medium was replaced with the seventh prepared medium.

(5) The transparent multiwell plate exchanged with the seventh preparation medium was placed on the LED irradiation apparatus in the CO ₂ incubator. The prepared transparent multi-well plates were irradiated with light under each light irradiation condition.

(6) Each multiwell plate was stored in a storage CO ₂ incubator immediately after light irradiation.

(7) In each multiwell plate stored in a CO ₂ incubator, osteoclasts cultured in each well 2 days, 4 days, 6 days, 8 days, 10 days, 12 days and 14 days after light irradiation Stained with a TRAP staining kit (AK04F) manufactured by Primary Cell.

2. Procedure for Experiment 3-2-2

(1) LONZA human osteoclast precursor cell basic medium OBPM, LONZA L-Glutamate so that the final concentration is 2 mM, LONZA FBS so that the final concentration is 10%, and so that the final concentration is 1% A fifth prepared medium supplemented with LONZA GA-1000 was prepared.

(2) The human-derived osteoclast precursor cells (2T-110) manufactured by LONZA were cultured at 37 ° C. in a fifth preparation medium in a CO ₂ incubator.

(3) Prepare a sixth conditioned medium in which LONZA RANKL is added to the fifth prepared medium so that the final concentration is 30 ng / ml, and LONZA M-CSF is added so that the final concentration is 10 ng / ml. Human-derived osteoclast progenitor cells were seeded in a multiwell plate equipped with a dentin slice, and replaced with the sixth preparation medium, and then cultured in the sixth preparation medium at 37 ° C. for 2 weeks in a CO ₂ incubator. did.

(4) A seventh prepared medium prepared by adding R & D SYSTEMS TNF-α to a final concentration of 10 ng / ml in the sixth prepared medium is prepared, and human-derived osteoclast precursor cells are cultured. The prepared medium was replaced with the seventh prepared medium.

(5) The transparent multiwell plate exchanged with the seventh preparation medium was placed on the LED irradiation apparatus in the CO ₂ incubator. The prepared transparent multi-well plates were irradiated with light under each light irradiation condition.

(6) Each multiwell plate was stored in a storage CO ₂ incubator immediately after light irradiation.

(7) In each multiwell plate stored in a CO ₂ incubator, the dentin slices of each well 4 days after light irradiation were collected, and the degree of dissolution of the dentin slices by osteoclasts was observed using a microscope. .

<Evaluation result of Experiment 3-2>
1. Evaluation Results of Experiment 3-2-1 FIGS. 10A and 10B show the measurement results of the number of osteoclasts stained in red by TRAP staining. FIG. 10 (a), was irradiated in mature osteoclasts the migration was induced into the active form by introducing a TNF-alpha light of each wavelength at a power density 1 mW / cm ² to 10 mW / cm ² 50 sec The time-dependent change of the number of osteoclasts stained with TRAP later is shown. FIG. 10 (b) shows a case in which mature osteoclasts whose transition to the active form was induced by introducing TNF-α were irradiated with light of each wavelength at a power density of 1 mW / cm ^{2 to} 10 mW / cm ² for 500 seconds. The time-dependent change of the number of osteoclasts stained with TRAP is shown.

In the results shown in FIGS. 10 (a) and (b), near-infrared light (840 nm) having a power density of 1 mW / cm ² is applied to mature osteoclasts in which transition to an active form is induced by introducing TNF-α. When irradiated for 50 seconds or 500 seconds [black squares in FIGS. 10A and 10B], when irradiated with near-infrared light (840 nm) having a power density of 10 mW / cm ² for 50 seconds or 500 seconds [FIG. (□ in (a) and (b)], when irradiated with red light (660 nm) having a power density of 1 mW / cm ² for 50 seconds or 500 seconds [black triangles in FIGS. 10 (a) and (b)], power When red light (660 nm) having a density of 10 mW / cm ² is irradiated for 50 seconds or 500 seconds [Δ in FIGS. 10A and 10B], blue light (470 nm) having a power density of 1 mW / cm ² is applied for 50 seconds. Or irradiated for 500 seconds Case [● in FIGS. 10 (a) and (b)] and blue light (470 nm) with a power density of 10 mW / cm ² was irradiated for 50 seconds or 500 seconds [circles in FIGS. 10 (a) and (b) In any of the cases, the number of osteoclasts stained with TRAP was smaller than in the case of the control [X in FIGS. 10 (a) and (b)].

That is, the results shown in FIGS. 10A and 10B show that mature osteoclasts are irradiated with near-infrared light (840 nm), red light (660 nm), and blue light (470 nm). This suggests that the cells further suppress the transition to activated osteoclasts that produce TRAP.

2. Evaluation Results of Experiment 3-2-2 FIG. 11 shows the calculation results of the dissolution area of dentin slices by osteoclasts. FIG. 11 shows the results on the 4th day from the start of cultivation of osteoclasts in dentin slices.

From the results of FIG. 11, the near-infrared light of TNF-alpha power density in mature osteoclasts the transition to the active form was induced by introducing a 1 mW / cm ² to 10 mW / cm ² a (840 nm) 500 seconds when irradiated, if the power density 1 mW / cm ² to 10 mW / cm ² of red light (660 nm) was irradiated for 500 seconds, the power density (1 mW / cm ² to 10 mW / cm ² of blue light (470 nm) to 500 seconds irradiation In this case, the dissolution area of the ivory heterogeneous section was small as compared with the control.

Confirmation in Experiment 3-2-1 of light irradiation that mature osteoclasts are inhibited from transitioning to the active form in the results of FIG. We were able to.

<< Experiment 3-3 >>
Experiment 3-3 measured the amount of cartilage matrix proteoglycan produced by chondrocytes, when the chondrocytes in which the production of cartilage matrix proteoglycan was suppressed by IL-1β were irradiated or not irradiated. The effect of light was evaluated.

The chondrocytes used in Experiment 3-3 were collected from a knee osteoarthritis patient (78 years old, female). In addition, the irradiation conditions of the chondrocytes in Experiment 3-3 were the same as in [Table 1] in Experiment 1.

In addition, the results of the experiment performed under the above 12 conditions were chondrocytes in which the production amount of the cartilage matrix proteoglycan was suppressed by IL-1β, and negative (hereinafter referred to as “negative control”) when light irradiation was not performed. ”, A control in which the amount of cartilage matrix proteoglycan produced by IL-1β was not suppressed, and the case where light irradiation was not performed was defined as positive (hereinafter referred to as“ positive control ”). Based on the evaluation.

<Procedure for Experiment 3-3>
Experiment 3-3 was performed according to the following procedure.

(1) Equinec BIO, INC. Fetal bovine serum (hereinafter referred to as “FBS”) in Sigma basic medium (Dulbecco's modified Eagle's medium, Sigma) so that the final concentration is 10%. And Penicillin-Sterptomycine (5000 U / ml) manufactured by GIBCO was added to a final concentration of 1% to prepare a first preparation medium.

(2) Chondrocytes collected from the patient were subcultured twice at 37 ° C. in the first preparation medium in a CO ₂ incubator.

(3) Prepare a second preparation medium in which IL-1β made by R & D SYSTEMS is added to the first preparation medium to a final concentration of 10 ng / ml, and prepare the first preparation medium in which chondrocytes are cultured. The medium was changed to the second preparation medium.

(4) As shown in FIG. 1, the transparent multiwell plate replaced with the second preparation medium was placed on the LED irradiation apparatus in the CO ₂ incubator. The prepared transparent multi-well plates were irradiated with light under each light irradiation condition.

(5) Each multiwell plate was stored in a storage CO ₂ incubator immediately after light irradiation.

(6) In each multiwell plate stored in a CO ₂ incubator, the culture supernatant of each well 24 hours after light irradiation was collected.

(7) The concentration of proteoglycan contained in the collected culture supernatant was measured by ELISA. For the proteoglycan concentration ELISA, a kit manufactured by DIA source Immuno Assays (DIA source Immuno Assays SA, Nivelles, Belgium) was used.

<Evaluation results of Experiment 3-3>
1. Evaluation Results of Experiment 3-3 FIG. 12 shows the measurement results of the amount of cartilage matrix proteoglycan produced 24 hours after irradiation of chondrocytes derived from knee osteoarthritis patients.

IL-1β (−) shown in FIG. 12 indicates a case where the amount of cartilage matrix proteoglycan produced by chondrocytes is not suppressed by IL-1β, and IL-1β (+) indicates that the chondrocytes are chondrocytes by IL-1β. The case where the production amount of the substrate proteoglycan is suppressed is shown. Further, the LED (−) shown in FIG. 12 indicates a case where the chondrocytes are not irradiated with light. Therefore, IL-1β (−) and LED (−) in FIG. 12 show the result of positive control, and IL-1β (+) and LED (−) show the result of negative control.

From the results of FIG. 12, it can be seen that the amount of cartilage matrix proteoglycan produced by chondrocytes is greater when irradiated with blue light (470 nm) than in the negative control.

That is, it can be said that the result of FIG. 12 suggests that the production of cartilage matrix proteoglycan from chondrocytes is promoted by irradiation with blue light (470 nm).

From the results of Experiment 3-3, the possibility that blue light (470 nm) has an effect of promoting the production of cartilage matrix proteoglycan in chondrocytes could be newly found.

<< Summary of Embodiment 1 >>
From the above experimental results, it was found that near-infrared light promotes bone formation while suppressing osteolysis. That is, it was found that near-infrared light may contribute to improving the balance of bone metabolism by suppressing bone resorption that is abnormally advanced by metabolic bone diseases such as rheumatoid arthritis.

On the other hand, blue light and red light resulted in suppression of osteolysis but also suppression of bone formation.

On the other hand, it was found that blue light has an effect of promoting the production of cartilage matrix proteoglycan in chondrocytes. That is, it has been found that blue light may be able to suppress cartilage degeneration and destruction seen in osteoarthritis and the like.

(Embodiment 2)
From the experimental results of the first embodiment, it has been found that near-infrared light may have an effect of improving the balance of bone metabolism that has been destroyed by metabolic bone disease. In Embodiment 2, a rat in which rheumatoid arthritis is artificially induced is used as a model, and it is shown that near-infrared light is useful for improving the balance of bone metabolism.

≪Experiment 4≫
Experiment 4 shows a method for creating a rat model in which rheumatoid arthritis is simulated. The model created in Experiment 4 is a model (hereinafter abbreviated as “CIA model”) that developed collagen-induced arthritis (hereinafter abbreviated as “CIA”). Has symptoms similar to those of the abnormal synovial growth characteristic of human rheumatoid arthritis. In each experiment in the second embodiment, the CIA model created in Experiment 4 was used.

The CIA model used in the second embodiment was created using a Lewis rat (LEW / CrlCrlj: Charles River), and was particularly compared with a healthy Lewis rat [FIG. 13 (a)]. It has a symptom that the hind limbs are greatly swollen [FIG. 13 (b)].

<Procedure of Experiment 4>
Experiment 4 was performed according to the following procedure to obtain a CIA model.

(1) 1 ml of Chondrex Icomplete Freund's Adjuvand 1 ml, Chondrex 2 mg / ml bovine type II collagen solution for immunization (0.05 M acetic acid) 0.8 ml and 16 mg / ml N-Acetylmuramyl-L-alanyl-D -A mixed solution in which 0.2 ml of isoglutamine hydrate was mixed was prepared, and the mixed solution was ice-cooled.

(2) The mixed solution cooled with ice was stirred for 90 seconds at about 30,000 revolutions with a mechanical homogenizer, and then further cooled with ice for 5 minutes. The procedure of (2) was performed 4 times, whereby an emulsified arthritis inducing drug was prepared.

(3) A 7-week-old female Lewis rat was anesthetized, and 0.25 ml of an arthritis-inducing agent was injected into the tail skin.

(4) Further, one week after the first injection of the arthritis-inducing agent, 0.13 ml of the arthritis-inducing agent was injected into the tail skin of Lewis rats.

(5) By the above operation, a CIA model in which the hind limbs were swollen severely about 3 weeks after the first injection of the arthritis-inducing drug was obtained.

≪Experiment 5≫
In Experiment 5, an experiment was conducted to confirm that irradiation with near-infrared light to the hind limbs of the CIA model has an inflammation-inhibiting effect. In Experiment 5, a first experiment (hereinafter referred to as “Experiment 5-1”) and a second experiment (hereinafter referred to as “Experiment 5-2”) were performed.

<About Experiment 5-1>
Experiment 5-1 evaluated that the irradiation of near-infrared light suppressed inflammation of the hind limbs of the CIA model using the phenotype of the CIA model. Here, two experiments were conducted, Experiment 5-1-1 and Experiment 5-1-2.

Experiment 5-1-1 examined whether near-infrared irradiation centered on the knee part of the hind limb of the CIA model could suppress swelling of the heel part of the hind limb of the CIA model. This is an experiment to evaluate by measuring the thickness. Experiment 5-1-1 confirms that inflammation of the hind limbs of the CIA model can be suppressed if the thickness of the hind limb portion of the CIA model is reduced by light irradiation.

Specifically, an experiment was performed on the CIA model under the conditions shown in Table 2, and the average value of the thickness of the hind limb part of each of the five CIA models was determined as a group irradiated with near infrared light and a group not irradiated with light. I got it. Then, based on the thickness of the hind limb before the onset of arthritis in each group of rats, the degree of swelling of the average thickness of the hind limb after the onset ((average thickness of the hind limb after onset-limb fistula before and after onset) The influence of near-infrared light was evaluated by calculating the average value of the thickness of the part) × 100 / the average value of the thickness of the limbs before and after the onset). In Experiment 5-1-1, the day when the degree of swelling exceeded 90% was defined as day 0, and irradiation of near red light on the hind limbs of the CIA model was started.

Experiment 5-1-2 uses the same CIA model as Experiment 5-1-1. From a different point of view from Experiment 5-1-1, near-infrared irradiation centered on the knee of the hind limb of the CIA model causes inflammation. It is an experiment to evaluate that the motor function is improved by suppressing the above. Specifically, in Experiment 5-1-2, the amount of behavior of the CIA model was analyzed with and without near-infrared irradiation centered on the knee leg portion of the CIA model. The analysis of the action amount of the CIA model is a calculation of the total travel distance of the CIA model for 5 minutes on each measurement day after the 0th day of Experiment 5-1-1. This total movement distance was calculated by capturing the behavior of the CIA model with an image and tracking the movement of the rat by image analysis.

<Evaluation results of Experiment 5-1>
1. Evaluation Results of Experiment 5-1-1 FIG. 14 shows the evaluation results of Experiment 5-1-1. As can be understood from FIG. 14, in the case of irradiation with near-infrared light (◯ in FIG. 14) and in the case of no irradiation with near-infrared light (● in FIG. 14), day 0 As a reference, it can be understood that the passage of the second and third days is the peak of swelling of the thickness of the hind limb portion of the CIA model. As can be understood from FIG. 14, it can be understood that the swelling peak is suppressed when the near-infrared light is irradiated as compared with the case where the near-infrared light is not irradiated. However, when a certain period of time has passed after reaching the peak of swelling, no significant difference was observed between the case where the near infrared light was irradiated and the case where it was not irradiated.

That is, this result seems to suggest that the irradiation of near infrared light to the hind limbs suppresses the peak of inflammation due to rheumatoid arthritis.

2. Evaluation Results of Experiment 5-1-2 FIG. 15 shows the evaluation results of Experiment 5-1-2. As can be seen from FIG. 15, the behavior amount of the CIA model irradiated with near-infrared light does not reach the behavior amount of the healthy rat as a control, but compared with the CIA model that was not irradiated, the near-infrared light It can be understood that the amount of behavior has remarkably recovered from the eleventh day after the start of irradiation.

FIGS. 16A to 16C show an example of the trajectory measurement result of the 5-minute action of the rat on the 25th day of the near infrared light irradiation in FIG. 16A shows a healthy mouse as a control, FIG. 16B shows a CIA model irradiated with near-infrared light, and FIG. 16C shows a movement locus of the CIA model not irradiated with near-infrared light. ing.

As can be seen from FIGS. 16A to 16C, it can be understood that the CIA model irradiated with infrared light clearly has a larger amount of behavior than the CIA model not irradiated.

That is, the results of FIGS. 15 and 16 (a) to 16 (c) show that, from the viewpoint of the phenotype of the CIA model, irradiation with near infrared light suppresses inflammation of rheumatoid arthritis and restores the motor function of the hind limb of the CIA model. It can be said that this suggests.

<About Experiment 5-2>
In Experiment 5-1, it was confirmed from the phenotype of the CIA model that near-infrared light irradiation had an inflammation-inhibiting effect. In Experiment 5-2, it was confirmed by pathological staining that irradiation with near-infrared light suppressed inflammation.

Specifically, in Experiment 5-2, the length of the knee joint of the right hindlimb of the rat on the 32nd day after irradiation with near infrared light that was stained with hematoxylin-eosin (hereinafter referred to as “HE staining”). The degree of synovial cell proliferation and the degree of erosion of joint cartilage and bone by synovial cells were evaluated by pathological specimens of axial sections.

<Evaluation result of Experiment 5-2>
17 (a) to 17 (c) show H.C. of a long-axis cross section of a typical rat right hind knee joint. E. Shows pathological specimen of staining. FIG. 17A is a healthy rat, FIG. 17B is a CIA model irradiated with near infrared light, and FIG. 17C is a long axis of the right hind knee joint of a CIA model not irradiated with near infrared light. H. of the cross section. E. Pathological specimen of staining. Further, the positions of the joint, synovium, cartilage, femur, and tibia in the pathological specimen of FIG. 17 are shown in FIG.

First, when comparing the results of pathological staining of the femur of a healthy rat [FIG. 17 (a)] and a CIA model [FIG. 17 (c)] that was not irradiated with near-infrared light, a plurality of the results shown in FIG. As shown by the position of the black triangle, it can be clearly understood that the synovial cells proliferate abnormally and erode the cartilage.

On the other hand, as shown in the position of the black triangle in the result of pathological staining of the femur of the CIA model irradiated with near-infrared light [FIG. 17 (b)], some synovial cells proliferated abnormally. Compared with the results of pathological staining of the femur of the CIA model that did not irradiate near-infrared light, although there was a part eroded by cartilage, the abnormalities of the synovial membrane were clearly observed by irradiation with near-infrared light. It can be understood that the growth is suppressed.

Based on the above results, MMP-3 shown in Experiment 1 in Embodiment 1, in which inflammation is suppressed by near-infrared light, and as a result, also lysis of cartilage by synovial cells induced by the inflammation is suppressed. In addition, the results reflecting the evaluation results of MMP-13 could be confirmed even when rats were used as models.

≪Experiment 6≫
In Experiment 6, it is shown that the bone metabolism balance is improved and the bone function is restored by irradiation of the CIA model with near-infrared light. As Experiment 6, three cases of Experiment 6-1, Experiment 6-2, and Experiment 6-3 were performed with or without near-infrared light irradiation to the CIA model.

In Experiment 6-1, an X-ray image of the short-axis cross section of the left limb femur of the CIA model was acquired when the near-infrared light was irradiated to the hind limb of the CIA model, and the short axis in the X-ray image was acquired. The brightness values of the cross sections were compared. Since the direct state of the bone density can be grasped from the X-ray image, the X-ray image was evaluated in Experiment 6-1.

Experiment 6-1 was also performed on the CIA model under the conditions shown in [Table 2] in the same manner as Experiment 5, and five CIA were used for each of the cases of irradiation with near-infrared light and those without irradiation. An X-ray image of the model was acquired. In addition, as in Experiment 5, the day when the swelling degree of the thickness of the hind limb portion of the CIA model exceeded 90% was defined as day 0, and irradiation of near-red light on the hind limb of the CIA model was started. An X-ray image of a short-axis cross section of the left limb femur of an eye CIA rat was acquired. Moreover, the X-ray image of the short-axis cross section of the left limb femur of three healthy rats was acquired as control.

In Experiment 6-2, it was evaluated that near-infrared light is useful for improving the balance of bone metabolism from the viewpoint of osteoblasts involved in bone formation in bone metabolism.

In Experiment 6-2, pathological staining of the epiphyseal site of the femur of the rat right hind limb was performed with and without near-infrared light irradiation. In Experiment 6-2, osteocalcin immunostaining using an anti-osteocalcin antibody was performed as a pathological stain. The reason for osteocalcin staining is that, as described above, osteocalcin is an osteogenesis marker, and that osteocalcin staining is confirmed in pathological specimens in many ways. This is because the activity of bone formation can be evaluated. That is, if the pathologic specimen when irradiated with near-infrared light is confirmed to have a lot of osteocalcin staining compared with the pathological specimen without irradiation, the irradiation with near-infrared light is caused by osteoblasts. It is shown that it contributes to activation.

In Experiment 6-3, it was evaluated that near-infrared light is useful for improving the balance of bone metabolism from the viewpoint of osteoclasts related to osteolysis in bone metabolism.

In Experiment 6-3, as in Experiment 6-2, pathologic staining using TRAP staining was performed using the epiphyseal site of the femur of the rat right hind limb. The reason for using TRAP staining is to evaluate whether or not near-infrared light suppresses differentiation of osteoclast progenitor cells because osteoclasts generate TRAP when osteoclasts start to differentiate as described above. Because it can.

<Evaluation result of Experiment 6>
1. Evaluation Results of Experiment 6-1 FIGS. 18 (a) to 18 (c) show X-ray images of the short-axis cross section of the left limb femur of a rat. 18A shows healthy rats set as controls (3 cases), FIG. 18B shows CIA models irradiated with near infrared light (4 examples), and FIG. 18C shows near infrared light irradiation. It is the X-ray image of the short-axis cross section of the left limb femur of the CIA model (5 cases) which was not performed.

As shown in FIG. 18A, the X-ray image of a healthy rat as a control is displayed in white up to the inside of the femur, so that it can be understood that the bone density is high. On the other hand, as shown in FIGS. 18 (b) and (c), the CIA model has a lower bone density because there are many portions displayed in black in the femur as compared with the control. it can.

Comparing the X-ray images of the CIA model irradiated with near-infrared light and the CIA model not irradiated with light, the inside of the femur of the CIA model irradiated with near-infrared light in FIG. 18B is displayed in black. It can be understood that there are few parts and the bone density is high.

As a result of taking the average of the luminance values inside the femur of these X-ray images, it was found that the bone density of the CIA model irradiated with near-infrared light was clearly high as shown in FIG. That is, the experimental result of Experiment 6-1 shows that the near-infrared light improves the balance of bone metabolism as shown in Embodiment 1 and promotes the recovery of rough bone from the state of bone. It can be said that it is a suggestion.

2. Results of Evaluation in Experiment 6-2 FIGS. 20A to 20C show the results of pathological staining of the epiphyseal site of the femur of the right hind limb of a typical rat. 20 (a) is a healthy rat set as a control, FIG. 20 (b) is a CIA model irradiated with near infrared light, and FIG. 20 (c) is a CIA model not irradiated with near infrared light. It is the result of pathological staining of the epiphyseal site of the femur. The positions of cancellous bone and epiphyseal cartilage in the pathological staining of FIGS. 20 (a) to 20 (c) are shown in FIG. 20 (a).

First, osteocalcin stained with an anti-osteocalcin antibody is a position indicated by a plurality of black triangles in FIGS. 20 (a) to 20 (c). When comparing the results of pathological staining of the epiphyseal site of the femur of the right hind limb of the healthy rat [FIG. 20 (a)] and the CIA model [FIG. 20 (c)] that was not irradiated with near infrared light, Although a plurality of osteocalcin stains were confirmed in the pathological specimen, the osteocalcin staining was not confirmed in the pathological specimen of the CIA model that was not irradiated with near infrared light. From this result, it was confirmed again that bone formation was suppressed by rheumatoid arthritis.

On the other hand, the pathologic stained specimen of the CIA model irradiated with near-infrared light [FIG. 20 (b)] was confirmed to have osteocalcin staining equivalent to that of healthy rats. That is, similar to the result shown in Experiment 2 of Embodiment 1, the result of improving osteoblast activity by near-infrared light and promoting bone formation was obtained.

3. Evaluation Results of Experiment 6-3 FIGS. 21A to 21C show the results of pathological staining of the epiphyseal site of the femur of the right hind leg of a typical rat. FIG. 21 (a) is a healthy rat set as a control, FIG. 21 (b) is a CIA model irradiated with near infrared light, and FIG. 21 (c) is a CIA model not irradiated with near infrared light. It is the result of pathological staining of the epiphyseal site of the femur.

First, the positions indicated by a plurality of arrows in FIGS. 21A to 21C are osteoclasts stained by TRAP staining. FIG. 21 shows that TRAP staining was observed in the pathological staining of the epiphyseal part of the femur of the right hind limb of the healthy rat, the CIA model irradiated with near infrared light, and the CIA model not irradiated with near infrared light. Bone cells are confirmed. That is, in Experiment 6-3, near-infrared light did not suppress osteoclast differentiation in the CIA model, and did not reflect the results of Experiment 3 of Embodiment 1.

<< Summary of Embodiment 2 >>
As described above, whether or not near-infrared light suppresses bone resorption cannot be determined from the experimental results of Embodiments 1 and 2, but at least near-infrared light has an effect of promoting bone formation, and balance of bone metabolism. It was found that there is a possibility of improving.

(Embodiment 3)
Embodiments 1 and 2 show that near-infrared light is useful for improving the balance of bone metabolism. Embodiment 3 shows that near-infrared light is useful for improving the balance of bone metabolism in patients with rheumatoid arthritis.

≪About phototherapy device for metabolic bone disease≫
The phototherapy device for metabolic bone disease shown in FIGS. 22 and 23 was used in an experiment for showing that near infrared light is useful for improving the balance of bone metabolism in patients with rheumatoid arthritis. The specific configuration of the phototherapy device for metabolic bone disease used in Experiment 7 will be described below.

FIG. 22 is a perspective view of the phototherapy device 1 for metabolic bone disease, in which the inside is seen through. FIG. 23 is a cross-sectional view in the XY direction of the phototherapy device 1 for metabolic bone disease of FIG. FIG. 24 is a diagram showing a usage state of the phototherapy device 1 for metabolic bone disease of FIG.

As shown in FIG. 22 and FIG. 23, the metabolic treatment for bone disease 1 is provided with an affected part insertion port 3 so that a hand which is an affected part can be inserted into an affected part insertion chamber 2 provided therein. Further, as shown in FIG. 23, a flat plate-like planar member 4 is fixed.

The planar member 4 is installed from the lower peripheral edge of the affected part insertion port 3 to the back side of the affected part insertion chamber 2. The phototherapy device 1 for metabolic bone disease is configured such that the hand 5 inserted into the affected part insertion chamber 2 from the affected part insertion port 3 can be placed on the planar member 4 as shown in FIG. . Hereinafter, the surface on which the hand 5 of the planar member 4 is arranged is referred to as a first surface 6. The planar member 4 is made of a material that transmits therapeutic light, and is a hard material such as a transparent acrylic resin or transparent glass.

As shown in FIG. 23, in the phototherapy device 1 for metabolic bone disease, a first light source unit 7 including a plurality of light sources (for example, LEDs) is opposed to the first surface 6 of the planar member 4. The first surface 6 is provided at a certain distance. And the 1st light source part 7 comprises a surface light source by outputting therapeutic light from each of several light sources, and becomes a structure which can carry out surface irradiation with respect to a hand.

In the metabolic bone disease phototherapy device 1, the second light source portion 8 having a plurality of light sources faces the second surface 9, which is the opposite surface of the first surface 6 of the planar member 4. However, the second surface 9 is provided at a certain distance. And the 2nd light source part 8 comprises a surface light source by outputting therapeutic light from each of several light sources, and becomes a structure which can carry out surface irradiation with respect to a hand.

The plurality of light sources of each of the first light source unit 7 and the second light source unit 8 outputs treatment light having near-infrared light as a main wavelength band having an effect of improving the balance of bone metabolism. As described in the first and second embodiments, since blue light and red light may suppress bone formation, the diseased part is finally irradiated from the phototherapy device 1 for metabolic bone disease. The treatment light preferably contains no blue light or red light component.

≪Experiment 7≫
Experiment 7 was performed using the phototherapy device 1 for metabolic bone disease having the above-described configuration. In Experiment 7, it was confirmed by MRI image diagnosis analysis that near infrared light improves the balance of bone metabolism by irradiating the hand of a subject who has developed rheumatoid arthritis with near infrared light.

<Experimental conditions>
1. About subjects In Experiment 7, as subjects, women (3 cases) 40 years of age or older who have been treated for 6 months or more after the start of anti-rheumatic drug treatment and whose medical condition is stable were targeted.

2. About irradiation conditions of near-infrared light The setting of the above-mentioned phototherapy device 1 for metabolic bone disease performed in Experiment 7 is that the irradiation power density of near-infrared light irradiated to the affected area as treatment light is 0.115 W. / Cm ² and the center wavelength was set to 850 nm.

3. About the treatment time and period of the near-infrared light hand In the experiment 7, the test subject was inserted into the affected part insertion chamber 2 via the affected part insertion port 3 of the above-described phototherapy device for metabolic bone disease 1 for 5 minutes. Treatment was performed by irradiating near infrared light. In one treatment, the right hand and the left hand were each irradiated with near-infrared light for 5 minutes each, and once a week for 8 weeks (a total of 9 treatments).

<Evaluation result of Experiment 7>
FIG. 25 is an MRI diagnostic image of the finger part before the near-infrared light irradiation and after the ninth treatment (8th week) in the first patient. In the MRI diagnostic image of the finger part before near-infrared light irradiation, as shown by the part surrounded by the broken-line circles in FIG. 25 in particular, there are many bone erosions and edema parts in the bone marrow that are black and unclear. An image was obtained. This indicates that bone is damaged by rheumatoid arthritis.

On the other hand, in the MRI diagnostic image of the finger part after the ninth treatment (8th week), especially as shown by the part surrounded by the broken circle in FIG. A white and clear image was obtained. That is, it was confirmed that the erosion of the phalange and the state of edema in the bone marrow were slightly improved by irradiating near infrared light.

FIG. 26 and FIG. 27 are MRI diagnostic images of the finger part of the second patient and the third patient, respectively, before near-infrared light irradiation and after the ninth treatment (8th week). This result also showed a tendency that the erosion of the phalange and the state of edema in the bone marrow were slightly improved by irradiating near infrared light as in the first patient.

<< Summary of Embodiment 3 >>
Although the experimental results of Embodiments 1 and 2 showed that near-infrared light is useful for improving the balance of bone metabolism, the bone state of the fingers was also obtained in experiments performed on actual subjects in Embodiment 3. From these results, it was found that near-infrared light is useful for improving the balance of bone metabolism.

According to the phototherapy device for metabolic bone disease of the present invention, the symptoms of metabolic bone disease itself can be improved.

Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit and scope of the invention. This application is based on a Japanese patent application (Japanese Patent Application No. 2014-004238) filed on January 14, 2014, and is incorporated by reference in its entirety.

DESCRIPTION OF SYMBOLS 1 Phototherapy device for metabolic bone diseases 2 Affected part insertion chamber 3 Affected part insertion port 4 Planar member 5 Hand 6 First face 7 First light source part 8 Second light source part 9 Second face

Claims (5)

A phototherapeutic device for metabolic bone diseases, comprising a light source that outputs treatment light having near infrared light as a main wavelength band, and at least a light source unit that irradiates the affected part with the treatment light output from the light source. The phototherapy device for metabolic bone disease according to claim 1, wherein the light source section includes a plurality of light sources, and the treatment light output from each of the plurality of light sources is irradiated onto the affected area. The phototherapy device for metabolic bone disease according to claim 1 or 2, wherein the light source unit irradiates the affected part with treatment light including blue light. The phototherapeutic device for metabolic bone disease according to any one of claims 1 to 3, wherein the near-infrared light is light having a wavelength of 700 to 900 nm. The joint bone according to any one of claims 1 to 4, wherein the metabolic bone disease is an articular bone or cartilage degenerative joint disease associated with any one selected from osteoporosis, Paget's disease, rheumatoid arthritis, and osteoarthritis. A phototherapy device for metabolic bone disease according to 1.

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