JP2020072974A - Cell activation method and cell activation device - Google Patents

Abstract

To provide an easily-manufacturable plasma device, and a method of its application.SOLUTION: A cell activation method for activating cells by irradiating plasma has an irradiation step, for supplying plasma generation gas between facing electrodes as much as 0.1 L or more and 5.0 L or less per minute, applying a voltage having a frequency of 0.5 kHz or more and less than 20 kHz to the plasma generation gas as much as 5.0 kVpps or more and less than 20 kVpps, and irradiating the cells with plasma. The irradiation step is performed twice or more at a prescribed interval.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell activation method and a cell activation device.

  Conventionally, an atmospheric pressure plasma generator is used for cleaning the surface of semiconductor substrates, glass substrates, various films, and the like. As a method of generating atmospheric pressure plasma, a method of applying an AC voltage between opposing electrodes to generate glow discharge plasma between the electrodes is generally used. Plasma processing by glow discharge is roughly classified into a direct method in which an irradiation target is set in a discharge space between electrodes and a remote method in which active species are blown from the discharge space to irradiate the irradiation target. The remote method is adopted when there is concern about electrical damage to the irradiation target.

  In the remote type plasma device, the electrode arrangement illustrated in FIG. 5 may be adopted. FIG. 5 is a schematic cross-sectional view of the plasma generation part of the conventional plasma device 100. The plasma device 100 includes a gas pipe 101 into which a gas for plasma generation is introduced from a lower end toward an upper end, a pair of electrodes 102a and 102b arranged on a side surface of the upper end of the pipe, the electrode and the tubular dielectric. And a nozzle 103 for accommodating the body. The gas pipe 101 is hollow and is an insulator. The gas released from the upper end of the gas pipe 101 is ionized between the pair of electrodes 102a and 102b (plasma generating unit) opposite to each other above the upper end to generate atmospheric pressure plasma. The atmospheric pressure plasma is irradiated from the irradiation port of the nozzle 103 to the outside.

  Since atmospheric pressure plasma emitted from a conventional plasma device has a high temperature, there is a problem that it is not suitable for irradiation of soft material containing water. For example, when a living body such as a cell, a living tissue, or an individual living body is irradiated, excessive heat stress is applied to the cell or tissue, which may cause a burn.

  For the purpose of solving the above problems, Patent Document 1 discloses an atmospheric pressure low temperature microplasma injection device. The apparatus is provided with a large number of holes in the positive electrode of the plasma generation unit, and a voltage of 2 to 3 kV is applied between the positive electrode and the negative electrode that also serves as a gas pipe to generate a large voltage at a relatively low temperature of about 41 ° C. It is said that atmospheric pressure plasma can be jetted.

Patent No. 5225476

  However, since the positive electrode of the plasma device described in Patent Document 1 has a large number of fine holes having a diameter of 100 μm or less that generate plasma and transmit plasma, the electrode structure is complicated. When manufacturing this electrode, a fine patterning technique applied in the field of MEMS is required, and there is a problem that the manufacturing cost of the electrode increases.

  The present invention has been made in view of the above circumstances, and provides a plasma device that is easy to manufacture and a method of using the plasma device. Further, a nitrogen gas plasma applicable to medical use and a method of irradiating the nitrogen gas plasma are provided.

<1> A cell activation method for activating cells by irradiating plasma, comprising:
A plasma generating gas is supplied at a rate of 0.1 L or more and 5.0 L or less per minute between opposing electrodes, and a voltage having a frequency of 0.5 kHz or more and less than 20 kHz is applied to the plasma generation gas at 5.0 kVpss or more and less than 20 kVpps. And an irradiation step of irradiating the cells with the plasma,
A cell activation method, wherein the irradiation step is performed twice or more with a predetermined time interval.
<2> Opposing electrodes,
A power supply control unit that controls the voltage applied between the opposing electrodes to a frequency of 0.5 kHz or more and less than 20 kHz and a voltage of 5.0 kVpss or more and less than 20 kVpps;
And a dose control unit for controlling the amount of the plasma generation gas introduced between the electrodes to be 0.1 L or more and 5.0 L or less per minute,
A cell activation device, wherein a voltage is applied between the electrodes to generate plasma, and the cells are irradiated with the plasma twice or more with a predetermined time interval.

Further, the present invention has the following aspects.
[1] A tubular dielectric into which an atmospheric pressure plasma generating gas is introduced, and an internal electrode that extends in the axial direction of the tubular dielectric in the inner space of the tubular dielectric and has a coil shape or a shape having irregularities on the surface A plasma device comprising: an outer electrode extending along the inner electrode, the outer electrode being provided outside the tubular dielectric.
[2] The plasma device according to [1], wherein the external electrode has a tubular shape surrounding the outer peripheral portion of the tubular dielectric.
[3] The plasma device according to [1] or [2], wherein the atmospheric pressure plasma generating gas is nitrogen gas.
[4] The plasma device according to any one of [1] to [3], wherein the irradiation port for atmospheric pressure plasma has a size that can be inserted into a human oral cavity.
[5] The plasma device according to any one of [1] to [4], wherein the generated atmospheric pressure plasma is used for treatment or activation of cells, living tissues or living individuals.
[6] The plasma device according to any one of [1] to [5], further comprising a dose control device that controls a dose of the generated atmospheric pressure plasma.
[7] The method of using the plasma device according to any one of [1] to [6], wherein an alternating current of less than 20 kVpp and less than 20 kHz is applied between the external electrode and the internal electrode. , A method of using a plasma device, which comprises generating atmospheric pressure plasma.
[8] Controlling the amount of atmospheric pressure plasma emitted from the irradiation port to be less than 5.0 L / min by controlling the amount of the atmospheric pressure plasma generating gas introduced into the tubular dielectric. [7] The method of using the plasma device according to [7].
[9] At least one of the alternating current and the introduction amount of the atmospheric pressure plasma generation gas is controlled so that the temperature of the atmospheric pressure plasma is 40 ° C. or lower at a distance of 1 mm or more and 10 mm or less from the irradiation port. The method of using the plasma device according to [7] or [8].
[10] Nitrogen gas characterized by cleaning or activating the irradiated part or promoting healing of a wound or an abnormality in the irradiated part when irradiated to cells, biological tissues or individual organisms plasma.
[11] A method of irradiating cells, living tissues, or living individuals with nitrogen gas plasma to clean or activate the irradiated portion or to promote healing of a wound or abnormality in the irradiated portion. Irradiation method of nitrogen gas plasma (excluding the case where the living individual is a human).
[12] The method for irradiating nitrogen gas plasma according to [11], which comprises irradiating periodontal tissue or teeth with nitrogen gas plasma.
[13] The method for irradiating nitrogen gas plasma according to [11], which comprises irradiating the epithelial tissue with nitrogen gas plasma.

The plasma device of the present invention can be manufactured at a relatively low cost.
According to the plasma device of the present invention, low temperature atmospheric pressure plasma can be irradiated. A living body or a soft material can be irradiated with this atmospheric pressure plasma to clean the irradiated surface. Further, the plasma irradiation can treat or activate the living body.
According to the method of using the plasma device of the present invention, low-temperature atmospheric pressure plasma can be easily generated by applying a predetermined alternating current between the electrodes provided in the plasma generation unit.
According to the nitrogen gas plasma and the irradiation method thereof of the present invention, it is possible to clean or activate the irradiated portion or promote healing of a wound or abnormality in the irradiated portion.

It is a schematic diagram of the plasma generation part in one embodiment of the plasma device of the present invention. It is sectional drawing cut | disconnected by the xx line of FIG. 1 and FIG. It is sectional drawing cut | disconnected by the yy line of FIG. It is a schematic diagram of the plasma generation part in another embodiment of the plasma device of this invention. It is a schematic diagram which shows the structure of the plasma generation part in the conventional plasma apparatus.

<Plasma device>
FIG. 1 shows a main part of the plasma device 1 according to the first embodiment of the present invention.
The plasma device 1 includes a plasma generation unit 2 in which the atmospheric pressure plasma generating gas G is ionized to generate atmospheric pressure plasma P, and an irradiation port 6 for irradiating the atmospheric pressure plasma P toward the outside.

  The plasma generation unit 2 is installed in the tubular dielectric 3 into which the atmospheric pressure plasma generating gas G is introduced, and is installed in the inner space of the tubular dielectric 3, and extends in the axial direction of the tubular dielectric 3 (arrow D1). The inner electrode 4 is shaped like a ring, and the outer electrode 5 is provided outside the tubular dielectric 3 and extends along the inner electrode 4. The constituent material of the internal electrode 4 and the external electrode 5 is not particularly limited as long as it is a conductive material, and a metal used for a known electrode of a plasma device can be applied. A power source E that applies a voltage between both electrodes is connected to the inner electrode 4 and the outer electrode 5.

  A cylinder (not shown) for supplying the atmospheric pressure plasma generating gas G is connected to the first end (rear end) of the tubular dielectric 3 through a pipe. The constituent material of the tubular dielectric 3 is not particularly limited, and a dielectric material used in a known plasma device can be applied, and examples thereof include glass, ceramics, and synthetic resin. The lower the dielectric constant of the tubular dielectric 3, the better. The sectional shape of the tubular dielectric 3 is not particularly limited, and examples thereof include a circle, an ellipse, a quadrangle, and a hexagon.

  The cowling 7 houses the outer electrode 5, the tubular dielectric 3 and the inner electrode 4 in the inner space. With this configuration, it is possible to prevent an electric shock from being accidentally brought into contact with the external electrode 5 or the internal electrode 4 from the outside. The cowling 7 is preferably made of an insulating material.

  The shape of the tip of the cowling 7 is tapered, and the irradiation port 6 for leading out the atmospheric pressure plasma P is opened at the top thereof. The irradiation port 6 is located above the tip of the tubular dielectric 3 and on the extension of the axis of the tubular dielectric 3.

  FIG. 2 shows a sectional view taken along line xx of FIG. The cylindrical cowling 7, the cylindrical outer electrode 5, the tubular dielectric 3, and the coiled inner electrode 4 are concentrically arranged from the outside toward the center in this order. The external electrode 5 is arranged in close contact with the outer peripheral surface of the tubular dielectric 3. The internal electrode 4 is arranged at a predetermined distance from the inner peripheral surface of the tubular dielectric 3.

FIG. 3 shows a sectional view taken along the line yy of FIG. However, the cowling 7 is omitted and not shown.
As shown in FIG. 3, the longitudinal directions of the tubular dielectric 3, the external electrode 5, and the internal electrode 4 are along the same direction (direction of arrow D1). In this cross section, the outer peripheral surface of the coil of the internal electrode 4 faces the inner peripheral surface of the external electrode 5 with the tube wall 3a of the tubular dielectric 3 interposed therebetween, and there are a plurality of locations 4p adjacent to each other. The respective points 4p are separated from each other and arranged in a dispersed manner.
With the above arrangement, the internal electrode 4 to which a voltage is applied is prevented from being excessively heated locally, and the low temperature atmospheric pressure plasma P is easily generated.

  The distance between each part 4p of the coil of the internal electrode 4 and the external electrode 5 may be the same or different. It is preferable that two or more of the plurality of adjacent portions 4p are close to the inner peripheral surface of the external electrode 5 at a distance that can generate atmospheric pressure plasma. The distance at which the low-temperature atmospheric pressure plasma can be easily generated is, for example, 0.01 to 2.0 mm.

The shape of the internal electrode 4 provided in the plasma device 1 is not limited to the coil shape, and may be a shape having irregularities on the electrode surface facing the external electrode 5. For example, a shape in which a plurality of warts (projections), grooves, holes, and through holes are formed on the outer peripheral surface of the rod-shaped or cylindrical internal electrode 4 can be given. The sectional shape of the internal electrode 4 is not particularly limited, and examples thereof include a circle, an ellipse, a quadrangle, and a hexagon.
Further, any other shape may be used as long as it is a shape that faces the inner surface of the outer electrode 5 at a plurality of locations on the outer peripheral surface of the inner electrode 4 in the cross section orthogonal to the length direction.

  FIG. 4 shows a schematic diagram of the plasma generator 2 of the plasma device 10 according to the second embodiment of the present invention. The same members as those of the plasma device 1 of the first embodiment are designated by the same reference numerals. On the outer peripheral surface of the internal electrode 4 ′ of the plasma device 10, a plurality of wart-shaped protrusions 4 q are arranged so as to be separated from each other and dispersed. The wart-shaped projections 4q are arranged as described above in the region (the region hidden in the drawing) which overlaps the external electrode 5 on the outer peripheral surface of the internal electrode 4'in FIG. FIG. 2 is a schematic cross section taken along the line xx in FIG.

As shown in FIG. 4, the longitudinal directions of the tubular dielectric 3, the external electrode 5, and the internal electrode 4 ′ are along the same arrow D1 direction. The wart-like protrusions 4q on the outer peripheral surface of the inner electrode 4'oppose the inner peripheral surface of the outer electrode 5 with the tube wall 3a of the tubular dielectric 3 interposed therebetween. There are a plurality of wart-shaped protrusions 4q that are close to the inner peripheral surface of the external electrode 5.
With the above arrangement, the internal electrode 4 ′ to which a voltage is applied is prevented from being excessively heated locally, and the low temperature atmospheric pressure plasma P is easily generated.

  The shape of the external electrode 5 is not particularly limited as long as it can be arranged along the internal electrode 4 (hereinafter, unless otherwise specified, it is not distinguished from the internal electrode 4 '), and examples thereof include a cylindrical shape, a rod shape, and a plate shape. A shape such as a shape can be used. Of these, a cylindrical shape is preferable, and a cylindrical shape having an inner diameter capable of being installed in close contact with the outer peripheral surface of the tubular dielectric 3 is more preferable. With such a cylindrical shape, the outer electrode 5 can be arranged so that the inner peripheral surface of the outer electrode 5 reliably faces the outer peripheral surface of the inner electrode 4.

  When the external electrodes 5 are rod-shaped or plate-shaped, the number of the external electrodes 5 to be installed is not particularly limited, and may be one or two or more. When two or more are installed, it is preferable that they are arranged on the outer periphery of the tubular dielectric 3 at equal intervals, because the ionized portions of the atmospheric pressure plasma generating gas G are dispersed.

  The shape of the cowling 7 is not particularly limited, and it is preferable that the outer electrode 5 and the inner electrode 4 can be housed in the inner space. Similarly, the tubular dielectric 3 is preferably housed in the internal space, but the tip of the tubular dielectric 3 may be projected outside the cowling 7. The cowling 7 preferably has an irradiation port 6 for irradiating the external plasma irradiation target with the atmospheric pressure plasma P emitted from the distal end portion of the tubular dielectric 3.

  The cowling 7 preferably functions as a nozzle for irradiating the atmospheric pressure plasma generated in the plasma generation unit 2 to the outside of the device. When the irradiation port 6 and the nozzle portion of the cowling 7 forming the irradiation port 6 are of a size that can be inserted into the human oral cavity, the atmospheric pressure plasma P can be easily applied to the purpose of dental treatment or aesthetic treatment.

  The plasma device 1 preferably includes a power supply controller (not shown) that controls the voltage and frequency of the power supply E that applies a voltage between the external electrode 5 and the internal electrode 4. Examples of the power supply control unit include known power supply control devices.

  The plasma device 1 preferably includes a dose control unit (not shown) that controls the dose of the atmospheric pressure plasma P. An example of the irradiation amount control unit is a device that controls the introduction amount of the plasma generating gas G introduced into the tubular dielectric 3. Specifically, for example, there is a known mass flow controller that controls the flow rate of the gas introduced into the tubular dielectric 3 from the cylinder (not shown) of the plasma generating gas G through the pipe.

  As described above, the plasma generator 2 of the plasma device 1 has a simple structure, and thus can be manufactured at a relatively low cost.

<How to use the plasma device>
The usage method of the plasma apparatus 1 of FIG. 1 is illustrated. The gas G for plasma generation supplied from the cylinder is introduced from the lower end (first end) of the tubular dielectric 3 to the inner space thereof. The plasma generating gas G introduced into the inner space is ionized at a plurality of locations 4p where the inner electrode 4 and the outer electrode 5 installed at the tip portion (second end portion) of the tubular dielectric 3 face each other, and the The atmospheric pressure plasma P is obtained. At this time, a voltage is applied between the inner electrode 4 and the outer electrode 5.

  Atmospheric pressure plasma P generated at a plurality of locations 4p where the outer peripheral surface of the inner electrode 4 and the inner peripheral surface of the outer electrode 5 face each other is emitted from the tip of the tubular dielectric 3 and is irradiated in the inner space of the cowling 7 with an irradiation port. You are guided to 6. The atmospheric pressure plasma P is irradiated with the irradiation port 6 directed toward the irradiation target. The inner space of the tip of the cowling 7 is tapered, and the irradiation port 6 is provided on the top thereof, so that the tip of the cowling 7 functions as a nozzle.

In the inner space of the tubular dielectric 3, the outer peripheral surface of the inner electrode 4 and the inner peripheral surface of the outer electrode 5 are arranged to face each other with the tube wall 3 a of the tubular dielectric 3 interposed therebetween. With this arrangement, a plurality of points 4p where the electric field concentration for ionizing the atmospheric pressure plasma generating gas G occurs are dispersed. As a result, the internal electrode 4 to which the voltage is applied is prevented from being excessively heated locally, and the low temperature atmospheric pressure plasma P is easily generated.
Not only when the internal electrode 4 has a coil shape, but when the internal electrode 4 has a plurality of irregularities formed on its surface, similarly, low-temperature atmospheric pressure plasma can be easily generated.

  On the other hand, in the conventional plasma device 100 of FIG. 5, the electrodes 102a and 102b are locally close to each other at one location on the tip of the gas pipe 101, and electric field concentration occurs locally, so that the electrodes are not generated when plasma is generated. There is a problem that the temperature of the atmospheric pressure plasma generated by overheating increases.

In the plasma device 1, the content of active species contained in the atmospheric pressure plasma P and the temperature of the atmospheric pressure plasma P are controlled by controlling the voltage and frequency applied between the electrodes 4 and 5 by a power supply control unit (not shown). Can be controlled easily.
As an example, by applying a controlled alternating current in the range of less than 20 kVpp and less than 20 kHz between the external electrode 5 and the internal electrode 4, it is possible to generate the atmospheric pressure plasma P at 40 ° C. or less, for example.

The AC voltage applied between the electrodes 4 and 5 is preferably 5.0 kVpp or more and less than 20 kVpp, more preferably 6.0 kVpp or more and less than 15 kVpp, and even more preferably 7.0 kVpp or more and less than 10 kVpp. Here, the unit “Vpp (Volt peak to peak)” representing the AC voltage is the potential difference between the highest value and the lowest value of the AC voltage waveform.
By setting the applied AC voltage to be less than the upper limit value of each of the above ranges, the temperature of the generated atmospheric pressure plasma P can be suppressed low. The atmospheric pressure plasma P can be easily generated by setting the upper limit of each range above.

The frequency of the alternating current applied between the electrodes 4 and 5 is preferably 0.5 kHz or more and less than 20 kHz, more preferably 1 kHz or more and less than 15 kHz, further preferably 2 kHz or more and less than 10 kHz, particularly preferably 3 kHz or more and less than 9 kHz, and particularly preferably 4 kHz or more and less than 8 kHz. Is most preferred.
By setting the frequency of the alternating current to be less than the upper limit value of each of the above ranges, the temperature of the generated atmospheric pressure plasma P can be suppressed low. The atmospheric pressure plasma P can be easily generated by setting the upper limit of each range above.

It is preferable to control the irradiation amount of the atmospheric pressure plasma P irradiated from the irradiation port 6 to less than 5.0 L / min by controlling the introduction amount of the plasma generating gas G into the tubular dielectric 3.
In the plasma device 1, the introduction amount of the plasma generating gas G and the irradiation amount of the atmospheric pressure plasma P correspond to each other in a ratio of approximately 1: 1. By controlling the introduction amount to be less than 5.0 L / min, the irradiation amount can be controlled to be less than 5.0 L / min.

The introduction amount and the irradiation amount are each preferably 0.1 L or more and less than 5.0 L per minute, more preferably 0.3 L or more and less than 3.5 L per minute, and further preferably 0.6 L or more and less than 2.0 L per minute. preferable.
When it is at least the lower limit value of each of the above ranges, the efficiency with which the atmospheric pressure plasma P acts on the irradiated surface is sufficiently enhanced.
When it is less than the upper limit of each of the above ranges, it is possible to prevent the temperature of the surface to be irradiated with the atmospheric pressure plasma P from rising excessively. Further, when the surface to be irradiated is wet, it is possible to prevent the surface to be irradiated from being rapidly dried. Furthermore, when the irradiated surface is the affected area, it is possible to prevent the patient from suffering pain.

The voltage and frequency of the AC power supply and the temperature and the frequency of the AC power supply are set so that the temperature of the atmospheric pressure plasma P irradiated from the irradiation port 6 becomes 40 ° C. or less at a position (irradiation distance) separated from the irradiation port 6 by a distance of 1 mm or more and 10 mm or less. It is preferable to control at least one of the introduced amounts of the atmospheric pressure plasma generating gas G.
For example, the temperature of the atmospheric pressure plasma P at the above-mentioned position can be controlled to 40 ° C. or lower by setting the voltage and frequency described above in a suitable range or by setting the introduction amount in a suitable range described above. ..
The temperature of the atmospheric pressure plasma P at the above position is measured by irradiating the atmospheric pressure plasma P in the air through the irradiation port 6 and installing the tip of the rod-shaped thermocouple from the irradiation port 6 to the above position. ..

The type of the atmospheric pressure plasma generation gas G to be introduced into the tubular dielectric 3 is not particularly limited, and for example, other known plasma generation gases such as oxygen, helium, argon, etc. may be used conventionally as far as the present inventors know. Nitrogen gas which has never been used can be used.
As described above, the plasma generation unit 2 of the plasma device 1 can efficiently ionize the nitrogen gas at the plurality of locations 4p of the internal electrode 4 to easily generate the low-temperature nitrogen gas plasma.
The atmospheric pressure plasma generation gas G introduced into the tubular dielectric 3 may be one type of gas or a mixture of two or more types.

  When nitrogen gas plasma is generated, the volume of nitrogen gas contained in the atmospheric pressure plasma generation gas G introduced into the tubular dielectric 3 is preferably more than 50%, more preferably 70% or more, It is more preferably 90 to 100%. The type of the remaining gas component is not particularly limited, and, for example, air can be mixed with nitrogen to generate a nitrogen gas plasma.

<Use of atmospheric pressure plasma>
The atmospheric pressure plasma P generated in the plasma device 1 is preferably used for the purpose of irradiating living bodies such as cells, living tissues, and living individuals. By irradiating the living body with the atmospheric pressure plasma P, the living body can be treated or activated. For example, by irradiating the affected area having cuts, abrasions, burns, or other external wounds or other abnormalities with the atmospheric pressure plasma P, an effect of promoting healing of the external wounds and abnormalities can be obtained.

  When the atmospheric pressure plasma P is applied to an affected area with trauma or abnormality, it may be required to reduce the irradiation dose for the purpose of preventing pain to the patient. In this case, the irradiation amount of the atmospheric pressure plasma P irradiated from the irradiation port 6 is reduced by decreasing the introduction amount of the plasma generating gas G introduced from the rear end portion of the tubular dielectric 3 of the plasma device 1. You can

It may be required to further promote the healing by increasing the concentration of the active species contained in the atmospheric pressure plasma P. In this case, when the atmospheric pressure plasma P is irradiated, the irradiation port 6 is brought closer to a distance of 0.01 mm or more and 10 mm or less with respect to the irradiated portion, so that the atmospheric pressure plasma P containing a higher concentration of active species is irradiated. can do.
Since the temperature of the atmospheric pressure plasma P emitted from the plasma device 1 can be set to 40 ° C. or lower, the temperature of the irradiated portion may be excessively high even when the irradiation port 6 is brought close to the irradiated portion. Absent. Therefore, even when the irradiated portion is the affected area, it is possible to irradiate the affected area without causing burns.

The nitrogen gas plasma generated in the plasma device 1 has an effect of promoting healing of a wound or an abnormality. As shown in Examples described later, by irradiating cells, living tissues, or living organisms with nitrogen gas plasma, the irradiated portion is cleaned or activated, or a wound or abnormality in the irradiated portion is healed. can do.
Examples of the biological tissue include organs such as internal organs, epithelial tissue covering the surface of the body and the inner surface of the body cavity, periodontal tissue such as gingiva, alveolar bone, periodontal ligament and cementum, teeth and bone.

  When irradiating nitrogen gas plasma for the purpose of promoting healing of trauma or abnormality, the irradiation frequency, the number of times of irradiation, and the irradiation period are not particularly limited. For example, nitrogen gas is irradiated at a dose of 0.5 L or more and 5.0 L per minute. When irradiating the affected area with plasma, irradiation conditions such as 1 to 5 times a day, 10 seconds to 10 minutes each time, 1 to 30 days are preferable from the viewpoint of promoting healing.

(Temperature measurement)
Using the plasma device 1, an alternating current of 8.2 kVpp and 7 kHz was applied between the outer electrode 5 and the inner electrode 4 to generate an atmospheric pressure nitrogen gas plasma. Nitrogen gas plasma was irradiated at 1 L / min toward the tip of a rod-shaped thermocouple installed at a distance of 2 mm or 10 mm from the irradiation port 6, and the temperature was measured.
As a result, it was about 36 ° C. at a distance of 2 mm from the irradiation port 6 and about 32 ° C. at a distance of 10 mm from the irradiation port 6.

(Promotion of wound healing 1)
Prepare a rabbit with two wounds on the back, and apply nitrogen gas plasma (8.2kVpp, 7kHz, 1L / min 40 ℃) at atmospheric pressure from a distance of about 5mm for one wound only. Was irradiated for 90 seconds. As a result, a scab was formed on the surface of the wound one day after the irradiation, and the area of the wound was reduced three days after the irradiation, and the healing was progressing. On the other hand, the remaining one wound, which was not irradiated with nitrogen gas plasma for comparison and control, showed no change after 1 day, and scabs began to be formed after 3 days.

When the cell tissue was observed 7 days after the wound irradiated with the above-mentioned nitrogen gas plasma, it was confirmed that the thick epidermis was regenerated, the proliferation of fibroblasts and the formation of blood vessels.
On the other hand, when the cell tissue of a comparative control wound that was not irradiated with nitrogen gas plasma was observed 7 days later, thin epidermis was regenerated, but neither fibroblast proliferation nor vascularization was observed.

(Promotion of wound healing 2)
Another rabbit with two wounds on the back was prepared, and only one wound was irradiated with nitrogen gas plasma at atmospheric pressure similar to the above once a day for 7 consecutive days. .. Observation of the cell tissue 7 days after the wound confirmed that the epidermis was thicker than when it was irradiated only once, that there was vigorous proliferation of fibroblasts, and that there were many neovascularizations. It was
On the other hand, when the cell tissue of a control wound that was not irradiated with nitrogen gas plasma was observed 7 days later, the thin epidermis was regenerated, but the proliferation of fibroblasts was little and no neovascularization was observed. ..

(Promotion of wound healing 3)
A normal rat (SD rat) having three wounds on the back was prepared.
Immediately after injuring the first wound, nitrogen gas plasma (8.2 kVpp, 7.4 kHz, 1 L / min, 35 ° C.) at atmospheric pressure was irradiated once for 90 seconds from a distance of about 1 mm. did.
The second wound was irradiated once immediately after the wound and then once a day for 5 days, for a total of 5 times of nitrogen gas plasma irradiation under the same conditions as above.
The third wound was not exposed to nitrogen gas plasma.
Comparing the conditions after 5 days from the wounded day, the healing progressed in the order of the second wound (daily irradiation), the first wound (only one irradiation), and the third wound (no irradiation). I was there.
From the above results, it is considered that irradiation with nitrogen gas plasma promoted wound healing.

(Promotion of wound healing 4)
Using diabetic rats (SDT Fatty), the same test as the above-mentioned normal rats (SD rats) was performed. As a result, similar to the above, it was confirmed that the healing proceeded in the order of the second wound (daily irradiation), the first wound (only once irradiation), and the third wound (no irradiation).

  From the above results, it is clear that irradiation of nitrogen gas plasma at atmospheric pressure promotes wound healing.

(Removal of biofilm)
For extracted human molars, remove tartar and dirt using an ultrasonic scaler, mechanically polish the tooth surface with a tooth surface abrasive, remove the abrasive etc. using an ultrasonic cleaner, Experimental tooth material was obtained by sterilization with ethylene oxide gas.
By attaching periodontopathic bacteria (P. Gingivalis) to the surface of the tooth material and culturing, a biofilm (plaque) was formed over a wide area of the surface of the tooth material.
The biofilm clinging to the surface of the tooth material was irradiated with a nitrogen gas plasma at atmospheric pressure (8.2 kVpp, 7.4 kHz, 1 L / min, 35 ° C.) for 90 seconds from a distance of about 1 mm.
After rinsing the tooth material after irradiation lightly in water and observing the tooth surface, the biofilm in the irradiated area was washed off. On the other hand, the biofilm on the part not irradiated with the nitrogen gas plasma remained attached.

  When a comparative experiment in which a mere nitrogen gas was blown instead of the nitrogen gas plasma was performed on the tooth material to which the biofilm was attached, the biofilm could not be removed.

(Treatment of periodontal disease 1)
The periodontal disease bacteria were infected around the first molar of a healthy hamster, and it was confirmed by the PCR test at a later date that the periodontal disease bacteria were proliferated. The inflamed gingiva of this hamster was irradiated with a nitrogen gas plasma at atmospheric pressure (8.2 kVpp, 7.4 kHz, 1 L / min, 35 ° C.) for 90 seconds from a distance of about 1 mm.
Observation of the gingiva at the irradiated site one day later revealed that inflammation was clearly improved. On the other hand, the inflammation of the gingiva in the area not irradiated with nitrogen gas plasma was not improved.

(Treatment of periodontal disease 2)
The gingiva of a hamster having the same periodontitis as above was irradiated with nitrogen gas plasma (8.2 kVpp, 7.4 kHz, 1 L / min, 35 ° C.) at atmospheric pressure for 90 seconds from a distance of about 1 mm. After that, the irradiation was performed once a day for 7 days, and the nitrogen gas plasma was irradiated 7 times in total under the same conditions as above.
Seven days after the first irradiation, the gingiva in the irradiated area was observed, and it was found that the inflammation had subsided and healed. On the other hand, the inflammation of the gingiva in the area not irradiated with the nitrogen gas plasma was hardly improved.

(Treatment of periodontal disease 3)
Surrounding the molars of four healthy beagle dogs (body weight: about 10 kg) by winding a thread around the upper left molars, feeding them with soft food, and raising them for 21 days in a state where stains tend to adhere to the surrounding molars. In addition, palpation caused gingivitis to the extent that bleeding did not occur.

About 2 of the above 4 beagle dogs suffering from gingivitis, nitrogen gas plasma at atmospheric pressure (8.2 kVpp, 7.4 kHz, per minute) was applied to the inflamed gingiva from a distance of about 1 mm. 1 L, 35 ° C.) for 90 seconds. This plasma irradiation was performed once every other day for a total of 7 times over 2 weeks.
Two weeks after the first irradiation, the gingiva at the irradiated site was observed, and it was found that the inflammation had subsided and healed.

About another one of the above four beagle dogs suffering from gingivitis, a simple nitrogen gas (1 L, 20 to 25 ° C.) was applied to the inflamed gingiva from a distance of about 5 mm for 90 minutes. Sprayed for seconds. This spraying was performed once every other day for a total of 7 times over 2 weeks.
Two weeks after the first spraying, when the gingiva at the sprayed site was observed, inflammation of the gingiva was not improved.

  The remaining one of the four beagle dogs suffering from gingivitis was untreated. When the gingiva was observed after 2 weeks, the inflammation of the gingiva was not improved.

(Activation of periodontal ligament cells)
Using a 12-well plate, human periodontal ligament cells were inoculated in a calcification medium (containing a reagent that stains red when the cells are calcified) and cultured by a known method.
The cultured cells in the three wells were irradiated with nitrogen gas plasma (8.2 kVpp, 7.4 kHz, 1 L / min, 35 ° C.) at atmospheric pressure for 90 seconds from above at a distance of about 1 mm. When the well was observed on the next day, red cells indicating calcification were darkly observed in a wide range in the well.
Nitrogen gas (1 L / min, 20 to 25 ° C.) was blown for 90 seconds from above at a distance of about 1 mm to the cultured cells in the other three wells. When the well was observed on the next day, red cells showing calcification were thinly observed in a part of the well.
No treatment was performed on the cultured cells in the other three wells. When the well was observed the next day, almost no red cells showing calcification were observed in the well.
From the above results, it is considered that the nitrogen gas plasma irradiation activates human periodontal ligament cells and promotes their calcification, whereby alveolar bone and teeth are joined and periodontal disease is improved.

  The present invention can be used in the medical field.

DESCRIPTION OF SYMBOLS 1 ... Plasma device, 2 ... Plasma generation part, 3 ... Tubular dielectric material, 3a ... Tube wall, 4 ... Internal electrode, 5 ... External electrode, 6 ... Irradiation port, 7 ... Cowling, P ... Atmospheric pressure plasma, G ... Large Atmospheric pressure plasma generating gas, E ... Power source, 100 ... Conventional plasma device, 101 ... Gas pipe, 102a ... Electrode, 102b ... Electrode, 103 ... Nozzle

Claims (2)

A cell activation method for activating cells by irradiating plasma,
A plasma generating gas is supplied at a rate of 0.1 L or more and 5.0 L or less per minute between opposing electrodes, and a voltage having a frequency of 0.5 kHz or more and less than 20 kHz is applied to the plasma generation gas at 5.0 kVpss or more and less than 20 kVpps. And an irradiation step of irradiating the cells with the plasma,
A cell activation method, wherein the irradiation step is performed twice or more with a predetermined time interval. Opposing electrodes,
A power supply control unit that controls the voltage applied between the opposing electrodes to a frequency of 0.5 kHz or more and less than 20 kHz and a voltage of 5.0 kVpss or more and less than 20 kVpps;
And a dose control unit for controlling the amount of the plasma generation gas introduced between the electrodes to be 0.1 L or more and 5.0 L or less per minute,
A cell activation device, wherein a voltage is applied between the electrodes to generate plasma, and the cells are irradiated with the plasma twice or more with a predetermined time interval.

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