Ulcer healing promotion device and combination for promoting ulcer healing on the body surface

Abstract

The present invention provides a device and combination for promoting the healing of ulcers on the body surface. [Solution] A body surface ulcer healing promotion device comprising a glucose concentration sensor 11 for detecting the glucose concentration in the exudate of an ulcer 2 on the body surface 1, vibrators 21A, 21B, and 21C for applying vibration to the ulcer 2, and a control device 31 for controlling the vibration of vibrators 21A, 21B, and 21C based on the glucose concentration in the exudate; and a combination for promoting ulcer healing comprising a vibrator for applying vibration stimulation to an ulcer on the body surface and its vicinity, and a dressing material positioned between the ulcer and the vibrator, wherein the vibration stimulation is performed by vibration of the vibrator via the dressing material.

Description

Technical Field

[0005] , , , , , ,

[0001] The present invention relates to treatment techniques, a body surface ulcer healing promotion device, and a combination for promoting ulcer healing.

Background Art

[0002] Diabetes and its complications are the main non-traumatic causes of lower limb amputations. In the majority, a foot ulcer develops before a lower limb amputation surgery. At the stage when a foot ulcer has developed, it is often difficult to correct hyperglycemia. Only two-thirds of foot ulcers ultimately heal, and the remaining ones are thought to result in some form of amputation surgery. Foot ulcers and amputation surgeries have a significant impact on the patient's life (see, for example, Patent Documents 1 to 3). As a treatment method for foot ulcers, there is local administration of platelet-derived growth factor.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] There are few options for treating ulcers on the body surface, and means for promoting ulcer healing while the patient goes through daily life are desired. Therefore, one object of the present invention is to provide a device for promoting the healing of ulcers on the body surface. The present invention is also directed to a combination for promoting ulcer healing.

Means for Solving the Problems

[0005] A device for promoting the healing of surface ulcers according to an aspect of the present invention comprises a glucose concentration sensor for detecting the glucose concentration in the exudate of a surface ulcer, a vibrator for applying vibration to the ulcer, and a control device for controlling the vibration of the vibrator based on the glucose concentration in the exudate.

[0006] In the above-described device for promoting the healing of surface ulcers, the ulcer may be an ulcer caused by diabetes.

[0007] In the above-described device for promoting the healing of surface ulcers, the ulcer may be a foot ulcer caused by diabetes.

[0008] The above-described device for promoting the healing of surface ulcers may be equipped with multiple vibrators.

[0009] In the above-described device for promoting the healing of surface ulcers, the control device may control the vibration of the vibrator so as to promote the production of arginine in cells.

[0010] In the above-described device for promoting the healing of surface ulcers, the control device may control the vibration of the vibrator so that the glucose concentration in the exudate becomes equal to or greater than the concentration that increases blood flow.

[0011] In the above-described device for promoting the healing of surface ulcers, the control device may vibrate the vibrator until the glucose concentration in the exudate falls below the concentration at which the amount of arginine produced by cells exceeds a threshold.

[0012] The above-described device for promoting the healing of surface ulcers may further include a blood flow sensor that detects blood flow near the ulcer, and a control device may control the vibration of the vibrator based on the blood flow.

[0013] In the above-described device for promoting the healing of surface ulcers, the control device may control the vibration of the vibrator so as to increase blood flow. Another aspect of the present invention relates to combinations for promoting ulcer healing, specifically the aspects described in [1] to [7] below. [1] A vibrator that applies vibrational stimulation to ulcers on the body surface and their vicinity, A dressing material located between the ulcer and the vibrator is provided. The vibration stimulation is performed by the vibration of the vibrator through the dressing material. A combination for promoting ulcer healing. [2] The vibrator according to [1], which gives a vibration stimulation with a vibration intensity of 500 mVpp or more and 2000 mVpp or less. [3] The combination according to [1], further comprising a vibration control device for controlling the vibration of the vibrator. [4] The combination according to [1], wherein the ulcer is an ulcer caused by diabetes. [5] The combination according to [1], wherein the ulcer is a foot ulcer caused by diabetes. [6] For activating AMP-activated protein kinase (AMPK) in the ulcer and adipocytes in the vicinity thereof. The combination according to any one of [1] to [5]. [7] For activating nitric oxide synthase in the ulcer and vascular endothelial cells in the vicinity thereof. The combination according to any one of [1] to [5]. [Advantages of the Invention]

[0014] According to the present invention, it is possible to provide a device for promoting the healing of ulcers on the body surface. Furthermore, according to the present invention, it is possible to provide a combination for promoting ulcer healing. [Brief Description of the Drawings]

[0015] [Figure 1] It is a schematic diagram showing a device for promoting the healing of body surface ulcers according to an embodiment. [Figure 2] It is a schematic diagram showing the relationship between hyperglycemia and metabolism according to an embodiment. [Figure 3] It is a schematic diagram showing the relationship between hyperglycemia and metabolism according to an embodiment. [Figure 4] It is a graph showing the uptake amount of 2-deoxyglucose according to the example. [Figure 5] It is a graph showing the uptake amount of 2-deoxyglucose according to the example. [Figure 6] It is a graph showing the uptake amount of 2-deoxyglucose according to the example. [Figure 7] It is a graph showing the uptake amount of 2-deoxyglucose according to the example. [Figure 8] It is a schematic diagram showing GLUT4 translocation according to the example. [Figure 9] It is a schematic diagram and a photograph showing GLUT4 translocation according to the example. [Figure 10] It is a photograph showing the presence or absence of GLUT4 translocation according to the example. [Figure 11] It is a graph of the ratio of the fluorescence intensity of GLUT4-derived fluorescence in the cell membrane to the fluorescence intensity of GLUT4-derived fluorescence in the cytoplasm according to the example. [Figure 12] It is a photograph of a rat according to the example. [Figure 13] It is a photograph of the wound of a rat according to the example. [Figure 14] It is a photograph of the wound of a rat according to the example. [Figure 15] It is a graph of the area of the wound of a rat according to the example. [Figure 16] It is a photograph of the tissue of the wound of a rat according to the example. [Figure 17] It is a photograph of the tissue of the wound of a rat according to the example. [Figure 18] It is a graph of the blood volume of a rat according to the example. [Figure 19] It is a circuit diagram of the vibration device according to the example. [Figure 20] It is a photograph of the wound of a rat according to the example. [Figure 21] It is a graph of the area of the wound of a rat according to the example. [Figure 22] It is a graph of the blood volume of a rat according to the example. [Figure 23] This is a photograph of the wound tissue of a rat in the example. [Figure 24] This is a graph showing the gene expression levels of rats in the example. [Figure 25] This is a graph showing the gene expression levels of rats in the example. [Figure 26] This is a graph showing the gene expression levels of rats in the example. [Figure 27] This is a photograph of a rat wound tissue stained with the anti-PTX3 antibody described in the example. [Figure 28] This graph shows the M1 / M2 macrophage ratio (the number of CD68-positive cells divided by the number of CD163-positive cells) in the example. [Modes for carrying out the invention]

[0016] As shown in Figure 1, the body surface ulcer healing promotion device according to this embodiment includes a glucose concentration sensor 11 for detecting the glucose concentration in the exudate of an ulcer 2 on the body surface 1, vibrators 21A, 21B, and 21C for applying vibration to the ulcer 2, and a control device 31 for controlling the vibration of vibrators 21A, 21B, and 21C based on the glucose concentration in the exudate.

[0017] The vibrators 21A, 21B, and 21C can be motors, pneumatic devices, transducer materials, piezoelectric foils, boil coils, and piezoelectric actuators. The number of vibrators 21A, 21B, and 21C in the body surface ulcer healing promoting device according to the embodiment is not particularly limited and may be one or multiple. For example, the vibrators 21A, 21B, and 21C are arranged to surround the patient's ulcer 2. The vibrators 21A, 21B, and 21C generate, for example, low-frequency vibrations.

[0018] Low frequencies are, for example, between 20Hz and 100Hz, between 30Hz and 90Hz, between 40Hz and 80Hz, between 40Hz and 70Hz, or between 40Hz and 60Hz, but are not particularly limited. The vibration intensity is, for example, between 100mVpp and 3000mVpp, between 500mVpp and 2500mVpp, between 500mVpp and 2000mVpp, or between 800mVpp and 2000mVpp, but are not particularly limited.

[0019] The vibrators 21A, 21B, and 21C may be placed on the body surface 1 via a dressing material 41. Examples of materials for the dressing material 41 include silicone, polyurethane, hydrophilic membrane, hydrophilic fiber, hydrocolloid, hydrogel, cellulose acetate, and polyvinyl alcohol. In other words, a configuration in which the vibrator is positioned on the body surface via a dressing material, namely a combination for promoting ulcer healing comprising a vibrator that applies vibration stimulation to an ulcer on the body surface and its vicinity, and a dressing material positioned between the ulcer and its vicinity and the vibrator, wherein the vibration stimulation is performed by vibration of the vibrator via the dressing material, is disclosed herein and is also the subject of the present invention.

[0020] The glucose concentration sensor 11 is positioned, for example, in contact with the patient's ulcer 2. The principle by which the concentration sensor 11 detects the glucose concentration is not particularly limited. For example, the glucose concentration sensor 11 comprises a membrane on which glucose oxidase is immobilized, a platinum electrode, and a silver electrode. When glucose is decomposed into gluconic acid and hydrogen peroxide by glucose oxidase, the hydrogen peroxide is oxidized at the platinum electrode and reduced at the silver electrode. The current generated at the electrodes is proportional to the glucose concentration. The glucose concentration sensor 11 detects the glucose concentration in the exudate of the ulcer 2 and transmits the glucose concentration in the exudate of the ulcer 2 to the control device 31.

[0021] The control device 31 controls the vibration of vibrators 21A, 21B, and 21C so that the vibration of the vibrators 21A, 21B, and 21C satisfies the conditions for promoting arginine production in cells. Examples of conditions for promoting arginine production in cells include vibration intensity and frequency.

[0022] The control device 31 may store a previously acquired relationship between the glucose concentration in the exudate and the amount of arginine produced intracellularly. The control device 31 may also store a previously acquired threshold value for the amount of arginine produced intracellularly necessary for the proper synthesis of nitric oxide. Generally, there is a negative proportional relationship between the glucose concentration in the exudate and the amount of arginine produced intracellularly. Therefore, a high glucose concentration in the exudate results in low arginine production, while a low glucose concentration in the exudate results in high arginine production.

[0023] The control device 31 controls the vibration of the vibrators 21A, 21B, and 21C so that the glucose concentration in the exudate detected by the glucose concentration sensor 11 is below the concentration at which the amount of arginine produced in cells exceeds a threshold. The control device 31 may also vibrate the vibrators 21A, 21B, and 21C until the glucose concentration in the exudate detected by the glucose concentration sensor 11 is below the concentration at which the amount of arginine produced in cells exceeds a threshold.

[0024] The control device 31 may store previously acquired relationships between glucose concentration in exudate and blood flow. The control device 31 may also store previously acquired appropriate blood flow thresholds. Generally, there is a negative proportional relationship between glucose concentration in exudate and blood flow. Therefore, a high glucose concentration in exudate results in low blood flow, and a low glucose concentration in exudate results in high blood flow.

[0025] The control device 31 controls the vibration of vibrators 21A, 21B, and 21C so that the glucose concentration in the exudate detected by the glucose concentration sensor 11 is below the concentration at which blood flow exceeds a threshold. The control device 31 may also vibrate vibrators 21A, 21B, and 21C until the glucose concentration in the exudate detected by the glucose concentration sensor 11 is below the concentration at which blood flow exceeds a threshold.

[0026] The body surface ulcer healing promotion device according to the embodiment may further include a blood flow sensor 12 that detects blood flow in the vicinity of the ulcer. The principle by which the blood flow sensor 12 detects blood flow is not particularly limited. For example, the blood flow sensor 12 includes a light source and a light-receiving element. Laser light emitted from the light source toward the human body is scattered by red blood cells in the blood vessels. The blood flow sensor 12 receives the scattered light with the light-receiving element and detects blood flow from the frequency spectrum of the scattered light. The blood flow sensor 12 transmits the detected blood flow to the control device 31.

[0027] The control device 31 may control the vibration of the vibrators 21A, 21B, and 21C based on blood flow. For example, the control device 31 may control the vibration of the vibrators 21A, 21B, and 21C to satisfy conditions for increased blood flow. Examples of conditions for increased blood flow include vibration intensity and frequency.

[0028] The ulcer may be a diabetic ulcer. The ulcer may be a foot ulcer. Normally, in the human body, glucose taken up by cells from the blood vessels is used for ATP production in glycolysis and the citric acid cycle. However, when glucose taken up by cells increases due to hyperglycemia, the glucose is taken up into the polyol metabolic pathway.

[0029] As shown in Figure 2, in the polyol metabolic pathway, glucose is converted to sorbitol by aldose reductase. When aldose reductase activity increases in the polyol metabolic pathway, the coenzyme NADPH (reduced nicotinamide adenine dinucleotide phosphate) is consumed. NADPH is also used in the synthesis of nitric oxide (NO) using arginine in vascular endothelial cells, but when NADPH is consumed in the polyol metabolic pathway, nitric oxide synthesis decreases. In addition, high glucose concentrations inhibit arginine production. When arginine production is inhibited, nitric oxide synthesis decreases. A decrease in nitric oxide leads to decreased blood flow and ischemia.

[0030] Although not bound by theory, it is believed that the body surface ulcer healing promoting device according to this embodiment activates AMPK (AMP-activated protein kinase) in adipocytes near ulcer 2 by applying vibration to ulcer 2, as shown in Figure 3, thereby improving the insulin resistance of adipocytes. Furthermore, it is believed that by improving the insulin resistance of adipocytes, the glucose concentration near adipocytes is optimized, nitric oxide is properly synthesized in vascular endothelial cells, and blood flow is promoted. Diabetic foot ulcers tend to form in areas with little muscle, such as the metatarsals and heels. In addition, diabetic patients also experience muscle atrophy due to peripheral neuropathy, resulting in reduced muscle function. Therefore, it is believed that the body surface ulcer healing promoting device according to this embodiment activates AMPK in adipocytes near ulcer 2. Another combination for promoting ulcer healing according to another embodiment of the present invention is a combination for activating AMP-activated protein kinase (AMPK) in the ulcer and adipocytes near it.

[0031] Furthermore, although not bound by theory, it is thought that mechanosensors in vascular endothelial cells promote the synthesis of nitric oxide synthase (NOS) in vascular endothelial cells in response to mechanical stress from the body surface ulcer healing promotion device according to the embodiment. Also, it is thought that activation of AMPK in adipocytes leads to the activation of nitric oxide synthase in vascular endothelial cells. In other words, the combination for promoting ulcer healing according to another embodiment of the present invention is a combination for activating nitric oxide synthase in vascular endothelial cells in the ulcer and its vicinity. It is thought that by promoting and activating the synthesis of nitric oxide synthase, nitric oxide is properly synthesized in vascular endothelial cells, and blood flow is promoted.

[0032] The body surface ulcer healing promotion device according to this embodiment can be miniaturized, allowing it to be permanently fixed to the patient's body surface while the patient maintains an independent daily life. Furthermore, a patient's insulin resistance is not constant but can fluctuate. The body surface ulcer healing promotion device according to this embodiment detects the glucose concentration in the exudate, which fluctuates due to insulin resistance, and controls the vibration of the vibrators 21A, 21B, and 21C, thereby suppressing unnecessary burden on the patient.

[0033] Ulcer healing is divided into two stages: the inflammatory phase and the proliferative phase. During the inflammatory phase, neutrophils and M1 inflammatory macrophages infiltrate the ulcer, causing edema. During the proliferative phase, fibroblasts migrate from the periphery of the ulcer, reconstructing the extracellular matrix, leading to angiogenesis and the formation of granulation tissue. In diabetic patients with chronic hyperglycemia, prolonged inflammation and an inability to progress to the proliferative phase are the reasons why foot ulcers are difficult to treat. The body surface ulcer healing promoting device according to this embodiment locally vibrates the vicinity of the ulcer to promote early wound contraction, thereby allowing the ulcer to progress to the proliferative phase early without prolonging inflammation.

[0034] (Example 1) 3T3-L1 adipocytes that had differentiated into adipocytes 8 days prior were prepared. The adipocytes were divided into two groups: one receiving vibration and the other not, and each group was cultured separately. The group receiving vibration was subjected to 50Hz vibrations at intensities of 600mVpp, 800mVpp, 1000mVpp, 1500mVpp, or 2000mVpp for 40 minutes daily.

[0035] After 7 days, as shown in Figure 4, the group that received vibrations had a higher uptake of 2-deoxyglucose by adipocytes than the group that did not receive vibrations. Furthermore, the uptake of 2-deoxyglucose by adipocytes was examined for 7 days. As a result, as shown in Figure 5, the uptake of 2-deoxyglucose by adipocytes increased over time when vibrations were applied.

[0036] When adipocytes were administered woltmannin, an insulin-dependent glucose uptake pathway inhibitor, woltmannin did not significantly inhibit the vibration-induced 2-deoxyglucose uptake promotion effect, as shown in Figure 6. On the other hand, when adipocytes were administered compound C, an AMPK inhibitor, compound C significantly inhibited the vibration-induced 2-deoxyglucose uptake promotion effect, as shown in Figure 7. This result suggests that the vibration-induced 2-deoxyglucose uptake promotion effect is due to the activation of AMPK by vibration.

[0037] (Example 2) Intracellular glucose uptake, facilitated by insulin and exercise, is carried out by the glucose transporter protein GLUT4. As shown in Figures 8 and 9, GLUT4 is normally stored in the GLUT4 storage compartment in the cytoplasm. When cells are stimulated by insulin or exercise, GLUT4 is transported from the storage compartment to the cell membrane. This phenomenon, called GLUT4 translocation, is known to be inhibited by insulin resistance in type 2 diabetes. GLUT4 translocation can be observed by immunofluorescence staining of GLUT4.

[0038] We observed whether GLUT4 translocation occurred in 3T3-L1 adipocytes after vibration at 50 Hz, 1000 to 1500 mVpp, 40 minutes / day for 5 days. As shown in Figure 10, in cells not treated with insulin or inhibitors, GLUT4 was present in the cytoplasm in untreated cells, but GLUT4 was eccentrically located at the cell membrane in treated cells. In cells treated with insulin but not with inhibitors, GLUT4 was eccentrically located at the cell membrane in both treated and untreated cells. In cells treated with woltmannin, GLUT4 was present in the cytoplasm in untreated cells, but GLUT4 was eccentrically located at the cell membrane in treated cells. In cells treated with Compound C, GLUT4 was present in the cytoplasm in both untreated and treated cells.

[0039] Figure 11 shows a graph of the ratio of fluorescence intensity in the cell membrane to fluorescence intensity in the cytoplasm. In cells treated with Compound C, no significant difference was observed between cells that were not subjected to vibration and cells that were. In cells treated with Waltmannin, a significant difference was observed between cells that were not subjected to vibration and cells that were. This result also suggests that the effect of vibration on promoting 2-deoxyglucose uptake is due to the activation of AMPK by vibration.

[0040] (Example 3) Seven-week-old male SD rats were prepared. The rats were given 55 mg / kg of streptozotocin. Rats that were administered intraperitoneally and whose blood glucose levels were 300 mg / dL or higher after 7 and 14 days were selected as a model rat for diabetes.

[0041] As shown in Figure 12, a 2 cm diameter full-thickness defect was created on the flank of an anesthetized diabetic model rat. A dressing (Foamlite, ConvaTec) was applied to the wound, and the trunk was secured with gauze. The dressing was changed daily.

[0042] Subsequently, for 14 days, a vibrator was placed on gauze over the wound, and local low-frequency vibrations were applied to the wound for 40 minutes each day while diabetic model rats were sedated with isoflurane inhalation anesthesia. The vibration period was 50 Hz, and the vibration intensity was 0 mVpp, 300 mVpp, 600 mVpp, or 1000 mVpp.

[0043] Observation of the wound appearance over time revealed that, as shown in Figures 13, 14, and 15, the wound contracted faster in diabetic model rats that were subjected to vibration than in diabetic model rats that were not subjected to vibration. Seven days after wound formation, as shown in Figure 16, observation of the wound tissue by hematoxylin-eosin staining revealed that vasodilation was more pronounced in diabetic model rats subjected to 1000 mVpp vibration than in diabetic model rats that were not subjected to vibration. Even 14 days after wound formation, as shown in Figure 17, vasodilation was still more pronounced in diabetic model rats subjected to 1000 mVpp vibration than in diabetic model rats that were not subjected to vibration.

[0044] (Example 4) In both non-diabetic rats and diabetic model rats, anesthesia was administered and wounds were formed in the same manner as in Example 3. The total tissue blood volume during anesthesia, during wound formation under anesthesia, and after waking from anesthesia were measured using a laser tissue blood oxygen monitor (OMEGAMONITOR, BOM-L1 TR SF, OMEGAWAVE, INC.). As shown in Figure 18, in both non-diabetic rats and diabetic model rats, the total tissue blood volume was higher upon waking than during anesthesia. This result suggests that changes in total tissue blood volume can be measured more accurately upon waking than during anesthesia.

[0045] (Example 5) Using a C1034 small vibration motor (Shin Shikou Denki Co., Ltd.), a vibration exciter with a circuit equivalent to the circuit diagram shown in Figure 19 was fabricated. Similar to Example 3, a 2 cm diameter full-thickness defect was created on the flank of an anesthetized diabetic model rat. A dressing was applied to the wound of the anesthetized diabetic model rat, and the vibration exciter was attached on top of the dressing. For 7 days, the rats were subjected to vibrations of 50 Hz and 600 to 1000 mVpp from the vibration exciter for 40 minutes each day. The rats were not anesthetized and remained awake when the vibrations were applied. In the control group, the vibration exciter was attached to the rats, but the vibration exciter was not activated.

[0046] As a result, as shown in Figures 20 and 21, the rats subjected to vibration showed faster wound contraction. Figure 22 shows a graph of the rats' blood volume, with the blood volume of the control rat on day 0 after wound formation set to 1. The blood volume of the vibration-treated rats increased daily. Therefore, it was shown that vibration not only temporarily dilated blood vessels but also increased the rats' steady-state blood volume.

[0047] Furthermore, seven days after wound formation, as shown in Figure 23, when the wound tissue was observed by hematoxylin-eosin staining, vasodilation was observed in diabetic model rats that were subjected to vibration compared to diabetic model rats that were not subjected to vibration, and numerous neovascularizations were also observed. This result indicates that vasodilation and neovascularization are related to the steady-state blood volume of the rats. This indicates that it has led to an increase.

[0048] (Example 6) In rats subjected to vibration in Example 3, gene expression was examined on day 4. As shown in Figure 24, the gene marker NOS3 for eNOS, an enzyme involved in vasodilation and synthesis of NO, and the angiogenesis marker Vegfa were elevated with 1000 mVpp vibration. Furthermore, the decrease in Tnf-α suggests that inflammation was suppressed and vasodynamics were improved.

[0049] In rats subjected to vibration in Example 3, wound healing progressed by day 14, and as shown in Figure 25, inflammatory markers (Tnf-α, Ptx, Ccl2) were significantly reduced in the vibration-treated rats compared to the unvibrated rats. Furthermore, NOS3, which is related to vasodilation, was significantly increased in rats subjected to vibrations ranging from 300 mVpp to 1000 mVpp.

[0050] In rats subjected to vibration in Example 5, gene expression was examined on day 7, and as shown in Figure 26, it was confirmed that inflammatory markers were reduced compared to the control group that was not subjected to vibration.

[0051] (Example 7) Similar to Example 3, diabetic model rats were prepared, and a 2 cm diameter full-thickness wound was created on the flank of anesthetized diabetic model rats. A dressing (Foamlite, ConvaTec) was applied to the wound, and the torso was fixed with gauze. The dressing was changed daily. For 14 days thereafter, a vibrator was placed on the gauze over the wound, and while the diabetic model rats were sedated with isoflurane inhalation anesthesia, local low-frequency vibration was applied to the wound for 40 minutes each day. The vibration period was 50 Hz, and the vibration intensity was 0 mVpp or 1000 mVpp.

[0052] Fourteen days after wound formation, wound tissue was collected and fixed by immersion in a 10% formalin solution overnight at room temperature. The tissue was then dehydrated using G-Nox (Genostaff), a substitute for ethanol and xylene, and embedded in paraffin to create a paraffin block. Furthermore, the paraffin-embedded tissue was sectioned to a thickness of 3 μm to produce tissue sections.

[0053] The tissue sections were immersed in G-Nox for 5 minutes three times to remove paraffin. Next, the tissue sections were immersed in ethanol for 5 minutes three times to remove G-Nox. Subsequently, the tissue sections were washed with purified water for 5 minutes twice. Furthermore, the tissue sections were incubated in 3% hydrogen peroxide diluted with methanol for 30 minutes to inactivate endogenous peroxidase in the tissue sections. In addition, the tissue sections were placed in 0.01 mol / L citrate buffer (pH 6.0) and autoclaved at 121°C for 15 minutes to reactivate the antigens in the tissue sections.

[0054] Next, the tissue sections were washed with phosphate-buffered saline (PBS), and the tissue sections were reacted overnight with anti-Pentraxin 3 (PTX3) antibody (rabbit-polyclonal, 13797-1-AP, Novus Biological) diluted 100-fold with bovine serum albumin / phosphate-buffered saline. After that, the anti-PTX3 antibody that was not bound to the antigen was removed from the tissue sections, and the tissue sections were washed with PBS three times for 5 minutes each.

[0055] Furthermore, tissue sections were incubated with 1000-fold diluted horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin antibody (Jackson ImmunoResearch) at room temperature for 1 hour. Next, they were incubated in 0.05 mol / L Tris-hydrochloride diluted with 2% hydrogen peroxide. Tissue sections were reacted with 0.2 mg / mL of 3,3'-diaminobenzidine [DAB] (Wako Pure Chemical Industries) in a cirrhosis solution (pH 7.4) to visualize HRP, and the reaction was stopped with purified water. Counterstaining with hematoxylin was then performed. The tissue sections were dehydrated with ethanol, cleared with G-Nox, and then mounted with mounting medium.

[0056] Stained tissue sections were observed under a microscope (BZ-X800, Keyence). As shown in Figure 27, it was confirmed that the inflammatory marker PTX3 was significantly reduced in rats subjected to vibration compared to rats that were not subjected to vibration.

[0057] (Example 8) Tissue sections were prepared in the same manner as in Example 7. However, in this example, the endogenous peroxidase in the tissue sections was not inactivated. The tissue sections were washed with phosphate-buffered saline (PBS), and the tissue sections were reacted overnight with anti-CD68 antibody (CD68 / SR-D1 antibody (ED1), mouse monoclonal, NB600-985-0.025, Novus Biological) and anti-CD163 antibody (CD163, EPR19518, rabbit monoclonal, ab182422, Abcam), which were diluted 100-fold with bovine serum albumin / phosphate-buffered saline. CD68 is a marker of M1 macrophages that promote inflammatory responses. CD163 is a marker of M2 macrophages that suppress inflammatory responses.

[0058] Subsequently, unbound anti-CD68 and anti-CD163 antibodies were removed from the tissue sections, and the tissue sections were washed with PBS for 5 minutes three times. Furthermore, the tissue sections were incubated with 1000-fold diluted green fluorescent dye-labeled anti-rabbit IgG antibody (Alexa Fluor 488, registered trademark, donkey, #711-545-152, Jackson ImmunoResearch) and red fluorescent dye-labeled anti-mouse IgG antibody (Alexa Fluor 594, registered trademark, donkey, #715-585-151, Jackson ImmunoResearch) at room temperature for 1 hour. Next, the tissue sections were washed with PBS, the nuclei were stained with a blue fluorescent dye using DAPI, and the tissue sections were mounted.

[0059] Stained tissue sections were observed under a microscope (BZ-X800, Keyence). Cells emitting green fluorescence were CD163-positive cells. Cells emitting red fluorescence were CD68-positive cells. The number of CD68-positive cells and CD163-positive cells were measured. Furthermore, the value obtained by dividing the number of CD68-positive cells by the number of CD163-positive cells (M1 / M2 macrophage ratio) was calculated. A smaller M1 / M2 ratio indicates improved inflammation. As a result, as shown in Figure 28, the M1 / M2 ratio was significantly reduced in rats subjected to vibration compared to rats that were not subjected to vibration. Explanation of the symbols

[0060] 11... Glucose concentration sensor, 12... Blood flow sensor, 21... Vibrator, 31... Control device, 41... Dressing material

Claims

1. A vibrator that applies vibrational stimulation to ulcers on the body surface and their vicinity, The system comprises a dressing material positioned between the ulcer and its vicinity and a vibrator, The aforementioned vibration stimulation is caused by vibration of a vibrator via a dressing material. A combination for promoting ulcer healing.

2. The combination according to claim 1, wherein the vibrator is a vibrator that provides vibration stimulation with a vibration intensity of 500 mVpp or more and 2000 mVpp or less.

3. Furthermore, the combination according to claim 1 is further provided with a vibration control device for controlling the vibration of the vibrator.

4. The combination according to claim 1, wherein the ulcer is an ulcer caused by diabetes.

5. The combination according to claim 1, wherein the ulcer is a foot ulcer caused by diabetes.

6. To activate AMP-activated protein kinase (AMPK) in the ulcer and adipocytes in its vicinity, The combination described in any one of claims 1 to 5.

7. To activate nitric oxide synthase in the vascular endothelial cells of the ulcer and its vicinity, The combination described in any one of claims 1 to 5.

Images