JP2021091619A - Dental oral composition and composition for treating bacterial infection - Google Patents

Abstract

To provide a dental oral composition that is suitable as an agent to control the infection of an intractable infected root canal tooth, has excellent permeability to a tooth root canal, and used for removing a smear layer, bacteria, and biofilms.SOLUTION: A dental oral composition includes an antibacterial agent and nanobubbles, the surface of the nanobubble being charged positively. The surface tension of positive nanobubble water that has decreased due to inclusion of the nanobubbles causes an increase of permeation power, and acts synergistically with the antibacterial action of the antibacterial agent, so that the infection of a tooth and an oral cavity can be controlled.SELECTED DRAWING: Figure 3

Description

The present invention is used for dental caries treatment, demyelination / infected root canal treatment, periodontal disease treatment, restoration / prosthesis treatment, peri-implantitis treatment, etc. The present invention relates to a dental and oral composition containing an antibacterial agent that suppresses bacteria in an internal restoration / prosthesis and a positively charged nanobubble (hereinafter referred to as plus nanobubble), and a composition for treating bacterial infection.

In a super-aging society, maintaining dental health and function is essential for achieving healthy longevity. When nerves are removed (extracted) with deep tooth decay, apical periodontitis (infected root canal) may occur in which pus accumulates under the roots due to reinfection within about 20 years after removal (frequency 15). ~ 20%, 10 million cases per year), and about 25% of them become intractable and continue to have tooth extractions and fractures. Therefore, the present inventor has developed a "pulp regeneration treatment method for preventing apical periodontitis even after demyelination", and has already confirmed the safety in 5 clinical studies and suggested its effectiveness () Non-Patent Document 1). Furthermore, in the canine apical periodontitis model, the apical periodontal tissue and pulp have been successfully regenerated by performing the same treatment method as for the demyelinated tooth after eradication (Non-Patent Document 2). On the other hand, the etiology of refractory infected root canals is that E. faecalis (Enterococcus faecalis), which is resistant to calcium hydroxide preparations usually used for root canal patches, invades deep into dentinal tubules and tooth roots with complicated pores. It is presumed to form a biofilm (Non-Patent Document 3). Therefore, in the treatment of intractable infected root canals, it is important to develop a method for cleaning and patching the root canal and removing the chemical and physical irritation of the apical periodontium as much as possible.

Refractory infected root canals not only affect occlusion, but also have a great effect on the whole body of the elderly with weakened immunity as a chronic source of infection. If you are taking an osteoporosis drug, you may not be able to extract teeth due to the risk of jaw bone necrosis. In addition, the number of cases in which implants can be applied by extracting teeth decreases in middle-aged and elderly people. Therefore, controlling infection of intractable root canal teeth, avoiding tooth extraction and fracture, and recovering tooth function are important for maintaining health in a super-aging society.

The inventor of the present invention uses a dental nanobubble generator to advantageously permeate a drug into a target affected area by utilizing the excellent permeation-enhancing effect of nano-sized ultrafine bubbles (negatively charged nanobubbles). We have proposed a drug composition that can be used (Patent Document 1). However, we have found that there is an antibacterial agent that cannot eradicate bacteria in the root canal when used in combination with negatively charged nanobubbles (hereinafter referred to as minus nanobubbles) as a drug for controlling infection of intractable root canal teeth. Therefore, by using minus nanobubbles in combination with antibacterial nanoparticles, it is possible to exert an effect of enhancing penetration and removing the smear layer, and by repeating root canal cleaning and root canal treatment every 1 to 2 weeks, the inside of the root canal can be treated. Can be sterilized. However, there is a problem that several root canal treatments are required for complete root canal eradication (bacteria in the root canal fall below the detection limit).

International release WO2016 / 084780A1

Nakashima M., Iohara K., Murakami M., Nakamura H., Sato Y., Ariji Y., Matsushita K .: Pulp regeneration by transplantation of dental pulp stem cells in pulpitis: A pilot clinical study. Stem Cell Res Therapy Mar 9; 8 (1): 61, 2017. Masanori Fujita, Koichiro Anhara, Naoki Horiba, Katsuro Tachibana, Hiroshi Nakamura, Misako Nakajima: "Intra-root canal sterilization and pulp regeneration using nanobubbles and ultrasound in infected root canal teeth" Japanese Dental Conservation Journal. 57 (2) ): 170-179, 2014. Takashi Okiji: "Issues in Endodontics-Pathogenesis and Clinic of Refractory Apical Periodontitis-" Niigata Dent. J. 36 (2): 1-15, 2006

The present invention has been made in view of such a problem, and is more suitable as a drug for controlling infection of a refractory infected root canal tooth, to a root canal of a tooth capable of complete eradication of the root canal in a shorter period of time. Dental and oral compositions containing positively charged nanobubbles (hereinafter referred to as plus nanobubbles) and compositions for treating bacterial infections, which have better permeability and are used for removing smear layers, removing bacteria, and removing biofilms. The purpose is to provide.

The dental and oral composition agent suitable for cleaning and patching the root canal of a tooth according to the present invention has the following constitution.
A dental or oral composition containing an antibacterial agent and nanobubbles, wherein the nanobubbles are positively charged plus nanobubbles on the surface thereof.
The surface tension of water is reduced by the positively charged plus nanobubbles contained in the composition, and the permeability of water is increased. The permeability of plus nanobubble water acts synergistically with the antibacterial action of the antibacterial agent, so that the antibacterial agent can be distributed deep into the root canal of the tooth and infection in the tooth and oral cavity can be controlled. .. Further, by using plus nanobubbles whose surface is positively charged, a composition for dentistry and oral cavity having an excellent antibacterial effect can be obtained.

The plus nanobubbles preferably have a positive zeta potential, a concentration of 1 × 10 ⁶ to 1 × 10 ⁹ cells / mL, and an average bubble diameter of 10 to 300 nm.

The antibacterial agents include antibacterial nanoparticles, antibiotics, antibacterial agents, antifungal agents, antiviral agents, disinfectants, root tube enlargers, antiinflammatory agents, physiologically active substances that promote wound healing and tissue regeneration, and stem cell-derived secretome. -It is preferable to include any one or more of the group consisting of exosome and miRNA. Further, it is more preferable that the antibacterial agent is a cationic antibacterial agent.

A composition for treating bacterial infection in tooth root canals, tooth periodontium, tooth caries or tongue coating, which comprises the plus nanobubble-containing dental oral composition of the present invention.

The dental and oral composition of the present invention effectively removes the smear layer in the root canal of the tooth, removes bacteria and biofilm, and the pulp and apical periodontium by using an antibacterial agent and plus nanobubbles in combination. It is possible to promote anti-inflammatory, healing, and regeneration. Therefore, it is possible to provide a composition for treating bacterial infection, which can completely eradicate intractable infected root canal teeth, control bacterial infection, and promote healing and regeneration by cleaning and patching the root canal.

It is sectional drawing which shows typically the method of making the refractory infected root canal model in a dog tooth. It is sectional drawing which shows typically the root canal cleaning and patch treatment by the combined use of nanobubbles and antibacterial nanoparticles in a canal refractory root canal model. It is a graph which shows the time-dependent change of the number of bacterial colonies in a root canal after root canal washing and application of antibacterial nanoparticle nanobubble water in a canal refractory infection root canal model. (A) Antibacterial nanoparticle final concentration 0.006% (w / v) / 98% minus nanobubble water, antibacterial nanoparticle final concentration 0.006% (w / v) only, (B) Antibacterial nanoparticle final concentration 0. 006% (w / v) / 98% plus nano bubble water, plus nano bubble water only. Changes in bone resorption image of apical lesions due to root canal treatment of 98% nanobubble water and final concentration 0.006% (w / v) antibacterial nanoparticles in a canal refractory infected root canal model before the start of treatment and 2 months after treatment It is a figure which was inspected by CBCT 4 months after, and was analyzed by OsiriX. (A) Drawing substitute photograph showing changes in bone resorption image by CBCT 3 months after root canal cleaning and root canal treatment by combined use of antibacterial nanoparticles and plus nano bubble water before surgery, (B) antibacterial nano Only particles, (C) combined use of antibacterial nanoparticles and minus nanobubble water, (D) combined use of antibacterial nanoparticles and plus nanobubble water for root canal cleaning and patch treatment 2 to 4 months after bone resorption volume It is a graph comparing preoperatively and statistically. ^** P <0.01 Comparison with preoperative. A fluorescent stereomicrograph in which a fluorescent agent (tetracycline) is impregnated into the deep part of the dentinal tubule after washing with various agents of minus or plus nanobubble water for 5 minutes is shown. (A) No drug, (B) Antibacterial nanoparticles, (C) Chlorhexidine gluconate, (D) Benzalkonium chloride, (E) Cetylpyridinium chloride, (F) Doxycycline, (G) Tetracycline-No drug. (Upper) Distilled water, (Middle) Minus nano bubble water, (Lower) Plus nano bubble water It is a drawing substitute photograph of a scanning electron microscope image after washing the smear layer in a root canal for 5 minutes by using various chemicals of minus or plus nano bubble water in combination. (A) No drug, (B) Chlorhexidine gluconate, (C) Cetylpyridinium chloride, (D) No cleaning. (Upper) Distilled water, (Middle) Minus nano bubble water, (Lower) Plus nano bubble water It is a graph which statistically analyzed the number of ivory tubules after washing ( ^{the number of ivory tubules per 1 mm 2 of the dentin wall). *} P <0.05, ^** P <0.01 Comparison with no treatment. ^{# P <0.05, ## P <} 0.01.

The dental and oral composition of the present invention contains an antibacterial agent and plus nanobubbles. Preferably, as an antibacterial agent which is a component of a dental or oral composition, it promotes antibacterial nanoparticles, an antibiotic, an antibacterial agent, an antifungal agent, an antiviral agent, a disinfectant, a root canal enlarger, wound healing and tissue regeneration. Physioactive substances Includes any one or more of the group consisting of anti-inflammatory agents, bioactive substances that promote wound healing and tissue regeneration, and stem cell-derived secretome, exosome, and miRNA.

The antibacterial nanoparticles are cyanoacrylate polymers, that is, acrylate-based polymer particles, and have a high affinity for the surface layer of the sugar chain peptide on the bacterial wall. When antibacterial nanoparticles are adsorbed on bacteria, the synthesis of the bacterial wall is inhibited in that part. Therefore, bacteria have the characteristic of autolyzing without maintaining internal pressure. Since antibacterial nanoparticles are biodegraded, they are not accumulated and are highly safe, cannot withstand, and do not destroy the natural environment. The side chain structure of the monomer used as a raw material for the cyanoacrylate nanopolymer is a linear n-butyl group and is safe because it does not generate formaldehyde in the metabolic system. In addition, antibacterial nanoparticles have an antibacterial effect at a low concentration.

The average particle size of the antibacterial nanoparticles is preferably 10 to 2,000 nm, more preferably 50 to 1,000 nm, and even more preferably 100 to 500 nm from the viewpoint of inhibiting the synthesis of the bacterial wall. In the present invention, the average particle size means the mode diameter (most frequent particle size). Further, in the present invention, the range "A to B" indicates A or more and B or less.

The concentration (w / v) of the antibacterial nanoparticles contained in the dental or oral composition is preferably 0.0001 to 0.3%, more preferably 0.001 to 0.06%, and 0.002 to 0.01. % Is more preferable. In the present invention, the concentration of antibacterial nanoparticles is indicated by weight / volume percent (% (w / v)), that is, the weight (g) of antibacterial nanoparticles contained in the dental orally composition 100 (mL).

The antibacterial nanoparticles can be produced, for example, by the method described in Japanese Patent No. 4963221. The antibacterial nanoparticles produced by this method are porous. Therefore, it is possible to conjugate the drug inside the pores of the antibacterial nanoparticles. Examples of the drug to be conjugated to the antibacterial nanoparticles include antibiotics and antibacterial agents such as ampicillin, doxycycline, vancomycin, and levofloxacin. Examples include antifungal agents, antiviral agents, disinfectants, root canal enlargers, anti-inflammatory agents, bioactive substances that promote wound healing and tissue regeneration, and stem cell-derived secretome exosome miRNA.

By conjugating the antibacterial agent with the antibacterial nanoparticles, the antibacterial agent adheres to the bacterial wall due to the adsorptivity of the antibacterial nanoparticles, and effectively exerts an antibacterial action. However, as described above, since the antibacterial nanoparticles exert an antibacterial action by inhibiting the synthesis of the bacterial wall and autolyzing the bacteria, they can be used without conjugating the antibacterial agent.

Some antibacterial agents affect the properties of nanobubbles. However, by using the antibacterial agent in a state of being conjugated to the antibacterial nanoparticles, the influence of the antibacterial agent on the nanobubbles can be suppressed.

Examples of the method of conjugating the drug with the antibacterial nanoparticles include a method of immersing the antibacterial nanoparticles in an aqueous solution of the drug, a method of anionic polymerization of a cyanoacrylate-based monomer in the coexistence of the drug, and the like. The latter method of anionic polymerization is more preferable because the two methods exemplified above are simple and have a high conjugation rate.

The concentration of the drug to be conjugated to the antibacterial nanoparticles can be appropriately set according to the properties of the drug, the volume at the time of use, and the like. When the antibacterial agent is conjugated, it is usually about 0.1 to 0.8% by weight in 100% by weight of the antibacterial nanoparticles.

As antibacterial agents that are components of dental and oral compositions, in addition to the above antibacterial nanoparticles, antibiotics, antibacterial agents, antifungal agents, antiviral agents, disinfectants, root canal enlargers, wound healing and tissue regeneration are promoted. Examples include bioactive substances, anti-inflammatory agents, bioactive substances that promote wound healing and tissue regeneration, and stem cell-derived secretome exosome miRNA. Preferably, doxicycline hydrochloride hydrate, levofloxacin hydrate, chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, monoammonium glycyrrhizinate, acrinol, sodium hypochlorite, povidone iodine, stannous fluoride. , EDTA, citric acid, MTAD, Tetracrene, trypsin, chymotrypsin, CCR3 antagonist, ALK5 inhibitor, mesenchymal stem cell secretome exosome. More preferably, doxycycline hydrochloride hydrate, chlorhexidine gluconate, benzalkonium chloride, cetylpyridinium chloride.

Antibacterial agents are cationic antibacterial agents such as cetylpyridinium chloride, chlorhexidine chloride, benzethonium chloride, benzalkonium chloride, depotassium chloride, and chlorhexidine gluconate due to their ionic properties; and amphoteric antibacterial agents such as protamine and dodecyldiaminoethylglycine. It is classified into nonionic antibacterial agents such as triclosan, 3-methyl-4-isopropylmethylphenol, thymol, carbachlor, farnesol, bisabolol, and cineole. Among them, the viewpoint of effectively exerting an antibacterial effect in the oral cavity. Therefore, a cationic antibacterial agent is preferable. Other common antibacterial agents such as hinokitiol; sodium lauroyl sarcosine; l-menthol can also be used.

The inventor of the present invention has already used a dental nanobubble generator to remove nanobubbles from the smear layer (cut pieces mixed with bacteria generated during root canal enlargement cleaning in dentin dentin tubules). A patent application has been filed for the ability to remove E. faecalis biofilms artificially formed on the surface of apatite (Japanese Patent Application No. 2017-152594). In the specification relating to the patent application, it was clarified by an in vitro experiment of the extracted tooth that nanobubbles permeate the drug deeper than 1 mm in the dentinal tubule. In the following, when the positive or negative of the chargeability of the surface does not matter, it may be simply described as nanobubbles.

Nanobubbles suppress the growth of bacteria by the action of free radicals generated during crushing. Furthermore, the zeta potential, which is the potential of the "slip surface" where liquid flow begins to occur in the electric double layer formed around the fine particles (nanobubbles) in the solution, is related to the fluidity, cohesiveness, storage stability, etc. of the fine particles. It is thought that.

Nanobubbles are contained as ultrafine bubbles in a liquid or gel-like form, and a gas (air, carbon dioxide, nitrogen, oxygen, ozone, etc.) is introduced into the nano-sized bubbles. That is, nanobubbles have a high internal pressure and an electric charge, and can effectively suppress the growth of bacteria by an effective removal promoting action based on their surface characteristics, kinetic characteristics such as Brownian motion, and the like. As the gas contained in the nanobubbles, one kind or two or more kinds can be used. When two or more kinds of gases are used, for example, there are cases where a mixture of nanobubbles consisting of only gas A and nanobubbles consisting of only gas B is used, and cases where nanobubbles containing a mixture of gas A and gas B are used. There is also.

When the dental or oral composition of the present invention is a liquid preparation, the solution constituting the composition is preferably an aqueous solution. In the present invention, an aqueous solution containing a gas in a nanobubble state is referred to as nanobubble water, and a gel containing a gas in a nanobubble state is referred to as a nanobubble gel.

In nanobubble water, the pH of the aqueous solution is not particularly limited, but can be, for example, 5.00 to 7.00, from weakly acidic to neutral. The absolute value of the zeta potential of nanobubbles does not change significantly over time. Further, in nanobubble water, the hardness is not particularly limited, but can be set to, for example, 20 to 30.

The average bubble diameter (mode diameter, most frequent bubble diameter) of the nanobubbles is preferably 60 to 300 nm, more preferably 80 to 250 nm, and even more preferably 100 to 200 nm. By setting the bubble diameter (diameter, size) of the nanobubbles in the above range, an effective removal promoting action can be obtained, and a high antibacterial effect can be obtained when used in combination with an antibacterial agent. The smaller the bubble diameter of nanobubbles, the better the stability of long-term storage in general.

Since the diameter of nanobubbles is nano-sized, the internal pressure of nanobubbles increases due to the surface tension of the nanobubbles. Here, the internal pressure of the nanobubbles is generally obtained by the Young-Laplace equation with respect to the bubble diameter of the nanobubbles, and the nanobubbles used in the present invention have an internal pressure of about 3 to 300 atm. ing.

One of the reasons why nanobubbles having such a high internal pressure are stably present in an aqueous solution is the presence of charged ionic components around the nanobubbles. That is, it is presumed that as the bubble diameter decreases, excess ions are retained at the interface between the gas and the liquid, and the zeta potential increases, which is involved in suppressing the disappearance of nanobubbles. It is considered that the ionic component acts as an electrostatic action between nanobubbles or between nanobubbles and liquid, and the electrostatic action prevents the disappearance of nanobubbles and stabilizes the nanobubbles in the liquid.

The dental and oral composition of the present invention contains positively charged plus nanobubbles whose surface is positively charged. Unlike conventional bubbles, Plus charged nano-bubbles have the same function as charged spherical capacitors. By using nanobubbles whose surface is positively charged, it is possible to give an electrical shock to bacteria whose inside is negatively charged, so that a composition for dental or oral cavity having an excellent antibacterial effect can be obtained.

The zeta potential inside the bacterium is about -60 mV to -30 mV. In order to give an electrical shock to such bacteria, plus nanobubbles in which the zeta potential of the nanobubbles in water is positive are preferable. The zeta potential is preferably +5 mV to 150 mV, more preferably +20 mV to 150 mV, and even more preferably +30 mV to 150 mV.

The concentration of nanobubbles is generally indicated as the number of bubbles (bubbles) contained in a specified volume. In the present invention, 1 × 10 ⁶ to 1 × 10 ⁹ bubbles / mL is preferable, and 1 × 10 ⁷ to 1 × 10 ⁹ pieces / mL is more preferable, and 5 × 10 ⁷ to 1 × 10 ⁹ pieces / mL is even more preferable. Here, if the abundance of such nanobubbles becomes too small, the removal promoting action cannot be advantageously exerted.

The size of the nanobubbles, the concentration thereof, and the zeta potential as described above can be measured using a commercially available nanoparticle measuring device. For example, Shimadzu Corporation's nanoparticle distribution measuring device (SALD-7100), Nippon Quantum Design Co., Ltd.'s nanoparticle analyzer (Nanosite NS500), Microtrack Bell Co., Ltd.'s ZetaView, etc. Be done. In addition, as a zeta potential measuring device, Zetasizer Nano Z, which can be obtained from Spectris Co., Ltd., Marballoon Division, Zeta Potential Measuring System (ELSZ-2000Z), Otsuka Electronics Co., Ltd., Kyowa Surface Chemistry Co., Ltd. Zeta potential measuring device (ZC-3000) and the like. In the present invention, nanosite NS500 and ZetaView are used for measurement. Therefore, when the measured value differs depending on the measuring device, the measured value obtained by using Nanosite NS500 or an equivalent product and ZetaView or an equivalent product is used as the characteristic value of nanobubbles.

By the way, in the present invention, nanobubbles having a nano-sized bubble diameter can be formed by using various known nanobubble generators. In particular, nanobubbles are formed by releasing a predetermined gas under the control of the amount of gas permeated through a breathable film formed by generating a craze, which is an initial destruction phenomenon, in a polymer resin film. Devices (eg, described in Japanese Patent No. 3806008, Japanese Patent No. 5390212) are used advantageously.

The method for producing nanobubble water or nanobubble gel is not particularly limited, but is, for example, as follows. That is, at least, a tubular gas permeation device for arranging a breathable film formed by generating craze on a polymer resin film capable of limiting the amount of gas permeation in the gas permeation portion is installed in the tubular circulation path. As a result, a hydraulic pressure is adjusted using a pump in the gap formed by the difference between the outer peripheral diameter of the tubular gas permeation portion and the inner peripheral diameter of the tubular circulation path, and water or a gel-like fluid is introduced. At the same time, by adjusting the pressurized state and supplying the gas to the gas permeation portion of the gas permeation device, nano-sized fine bubbles are mixed into the water or gel-like fluid.

Further, in a gas atmosphere that becomes bubbles, the liquid finely divided into micrometer size is further crushed to generate charged nanobubbles surrounded by the liquid, which are generated by gravity, centrifugal force, electromagnetic force, etc. It is possible to produce a nanobubble dispersion liquid such as nanobubble water charged in a liquid such as water by collecting using the above.

By applying an electric field to the gas atmosphere and grounding the negative side, negatively charged nanobubbles can be generated, and by grounding the crushing member, positively charged nanobubbles can be generated.

The dental and oral composition of the present invention can be prepared by mixing a liquid or gel-like precomposition containing an antibacterial agent with a water liquid or gel solid containing plus nanobubbles. The dental and oral composition can be used in combination with various compositions other than antibacterial agents, such as cell extracts, cell culture supernatants, microbial fermentation products, plant extracts, and purified proteins. However, these are used as long as they do not interfere with the function of the dental or oral composition, particularly the bacterial infection control action by the combined use of nanobubbles and an antibacterial agent.

The dental and oral composition of the present invention can be used as an agent for promoting removal of oral deposits. Oral deposits are, for example, smear layers, plaques, biofilms or tongue coating.
An oral adhering substance removal accelerator for removing the smear layer needs to not only remove the smear layer but also maintain dentin strength. A part of the smear layer penetrates into the ivory tubule, and the smear layer that penetrates into the ivory tubule may be called a smear plug or a smear plug.
As a removal accelerator for oral deposits for removing plaque, biofilm or tongue coating, nanobubble gel, which is a gel containing nanobubbles, is applied. The viscosity of the nanobubble gel is not particularly limited, but is, for example, 450 Mpa · s or less. This is because if the viscosity of the nanobubble gel is too high, the fluidity is lowered and the convenience may be lowered.

In addition, the dental and oral composition of the present invention can be used as a root canal enlargement aid for enlarging a stenotic root canal in middle-aged and elderly people. In this case, the dental oral composition will further include a root canal enlargement cleaning agent. The root canal enlargement cleaning agent is not particularly limited, and examples thereof include EDTA, citric acid, MTAD, and Tetracrene.

The dental and oral composition of the present invention can treat bacterial infection in the root canal of a tooth by the synergistic action of an antibacterial agent and plus nanobubbles. Therefore, it is effective for cleaning and patching intractable infected root canals and removing chemical and physical irritation of the apical periodontium as much as possible, in the root canal of the tooth, in the periodontal tissue of the tooth, and in the tooth. It can be used as a composition for treating bacterial infection of caries or tongue moss.

[Example 1]
After general anesthesia was applied to a 1-year-old dog, the upper and lower premolars of the dog were demyelinated, and the dog was expanded to the apex at # 50-55. After alternating washing with a 5% sodium hypochlorite solution and a 3% hydrogen peroxide solution, the washing was further carried out with physiological saline. A cotton ball was placed at the root canal opening, and the root canal was left open for 1 month. Furthermore, after washing with physiological saline, the inside of the root canal was completely dried with a paper point, and the inside of the root canal was completely temporarily sealed with cement and resin. Four months later, it was confirmed that an infected root canal was prepared by apical transmission images on a dental CT (CBCT / cone beam CT) (Fig. 1).

In order to measure the number of bacteria in the root canal, # 55 sterilized paper points soaked in physiological saline were placed in the root canal for 1 minute to fish, and the liquid medium Pradia for simple culture examination of bacteria in the root canal was performed. Paper points were added to (Product name, manufactured by Showa Yakuhin Kako Co., Ltd.). Then, the cells were seeded on a blood agar medium Vital Media (product name, manufactured by Far East Pharmaceutical Co., Ltd.) by a serial dilution method, and the number of colonies was counted after anaerobic culture for 2 days.

When the dog's tooth was opened for 1 month after demyelination and closed for 4 months, a transmission image was observed at the apex on CBCT (see FIG. 1). In the HE-stained image, alveolar bone resorption and apical periodontal tissue destruction were observed, and inflammatory cell infiltration was observed. When the bacteria in the root canal were observed on the Pradia medium, the medium became turbid and showed a positive result. The number of bacteria was infinite. Therefore, it was shown that a refractory infected root canal model can be prepared by opening the root canal for 1 month and blocking it for 4 months after demyelination.

(Effect of sterilization in root canal by combined use of nanobubble water and antibacterial nanoparticles in canal refractory root canal model)
For the above-mentioned refractory infected root canal, first, the number of bacteria in the root canal was anaerobically cultured for 2 days by a serial dilution method, and then the number of colonies was counted. The root canal after fishing was washed alternately with 6% sodium hypochlorite and 3% hydrogen peroxide solution, for a total of 2 mL each, and the root canal was further washed with 5 mL of physiological saline. Subsequently, the inside of the root canal is dried with a sterile paper point, and 2 mL of minus nanobubble water or plus nanobubble water containing 0.006% w / v antibacterial nanoparticles at the final concentration is placed in the root canal of the left upper and lower jaw humerus. It was injected and washed for 2 minutes. The right upper and lower premolars were washed with only 2 mL of antibacterial nanoparticles having a final concentration of 0.006% w / v, or with only 2 mL of plus nanobubble water. Then, the left side was washed with 5 mL of minus nanobubble water or plus nanobubble water and 5 mL of physiological saline. The right side was washed with 5 mL of saline only. Completely dry the inside of the root canal with a paper point, soak the left side with antibacterial nanoparticle / nanobubble water similar to cleaning, and the right side with only antibacterial nanoparticles or nanobubble water in the paper point and insert it into the root canal, and apply the patch. Was performed and temporarily sealed (see Fig. 2).

After 1 to 2 weeks later, the second fishing bacterium was carried out, and then the washing and patching treatment were performed using nanobubble water and antibacterial nanoparticles, antibacterial nanoparticles alone, or nanobubble water only, as in the previous time. Furthermore, one to two weeks after that, a third fishing bacterium was carried out, and the same washing and patching treatment as the previous treatment was performed again. The same operation was performed several times. After anaerobic culture for 2 days, the number of colonies was measured and statistically processed.

The number of bacteria gradually decreased by washing and patching with a combination of minus nanobubble water and antibacterial nanoparticles (minus nanobubble nanoverticle), and the number of bacteria decreased below the detection limit by washing and patching 5 times. Almost no decrease was observed with antibacterial nanoparticles alone (nanoparticles) (Fig. 3A). On the other hand, the number of bacteria decreased sharply at the first time and fell below the detection limit at the second time due to cleaning and patching with the combined use of plus nanobubble water and antibacterial nanoparticles (plus nanobubbles / nanoparticles). However, there was almost no change in the number of bacteria with only plus nanobubbles (plus nanobubbles) (Fig. 3B). Therefore, it was shown that the antibacterial nanoparticles alone or the plus nanobubble water alone did not have a bacterial removing effect. From this result, it can be seen that the antibacterial effect of the combined use of antibacterial nanoparticles and nanobubble water is affected by whether the nanobubble surface of the nanobubble water is charged positively or negatively.

(Reduction of apical lesions by nanobubbles and antibacterial nanoparticles in a canal refractory root canal model)
CBCT examination was performed at the start of intractable root canal treatment and 2 to 4 months after the start of root canal treatment. The transmission image of the apex (FIG. 4A) was image-analyzed by the OsiriX program, and the volume was measured. The results were expressed as a volume ratio before / after the start.
As a result, when only antibacterial nanoparticles were used for root canal treatment, it was 1.2 after 2 months and 1.5 after 4 months, and the apical lesion was enlarged (Fig. 4B). When minus nanobubble water or plus nanobubble water is used in combination with antibacterial nanoparticles, significantly bone addition at the apex is significantly performed 3 or 4 months after the start of root canal treatment compared to before surgery. The apical transmission image (apical lesion) was reduced due to ( ^** P <0.01) (Fig. 4C, Fig. 4D).

From the results shown in FIGS. 3A to 3B and 4A to 4D, it is possible to obtain a dental oral composition having an excellent sterilizing effect on intractable infected root canals by using nanobubble water and antibacterial nanoparticles in combination. I found out. In addition, by using plus nanobubble water as nanobubble water in which antibacterial nanoparticles are used in combination, the number of bacteria in the root canal could be reduced to below the detection limit earlier than minus nanobubble water. This result suggests that positive nanobubbles with a positively charged surface have a higher sterilizing effect when used in combination with antibacterial nanoparticles than negative nanobubbles with a negatively charged surface.

[Example 2]
(Measurement of number concentration, particle size distribution, zeta potential of nanobubble water)
Four kinds of solutions [50% plus nanobubble water, 50% minus nanobubble water, final concentration 0.2% chlorhexidine gluconate and 50% nanobubble water mixture, 0.2% chlorhexidine gluconate] were prepared by 10 mL each. For the prepared solution, the number concentration and particle size distribution of hyperfine bubbles existing in water were measured by Nanosite NS500 and ZetaView. In the examples, as the plus nano bubble water, nano bubble water produced by a plus nano bubble generator (NFPNC00003α, activation brush type, manufactured by Ohira Laboratory Co., Ltd.) using air and pure water was used. As the minus nanobubble water, nanobubble water produced by using air and pure water with a dental nanobubble generator FOAMEST 8 (registered trademark, manufactured by NAC Co., Ltd.) was used. The% of nanobubble water indicates the ratio of the undiluted solution produced by the nanobubble generator in the diluted solution.

For the measurement by Nanosite NS500 and ZetaView, about 5 mL of the sample was sampled with a syringe and applied to the sample injection port for measurement. The results are shown in Table 1. In Table 1, 50% nanobubble water is referred to as NB, a mixture of chlorhexidine gluconate having a final concentration of 0.2% and 50% nanobubble water is referred to as CHX + NB, and chlorhexidine gluconate having a final concentration of 0.2% is referred to as CHX.

As a result, as shown in Table 1, in the mixture of chlorhexidine gluconate having a final concentration of 0.2% and 50% nanobubble water, no nanobubble attenuation from 50% plus nanobubble water was observed. Therefore, it was suggested that chlorhexidine gluconate does not affect the nanobubble properties and can be used in combination with nanobubble water. The measurement result of CHX containing no nanobubbles is that nano-sized particles are counted as impurities. The presence / absence of nanobubbles can be determined by taking the difference between the presence / absence of nanobubble mixing.

[Example 3]
(Nano bubble water penetrates root canal dentin tubules)
The pseudo-root canal of the anterior tooth of the canal extracted tooth was expanded to # 60, and the apex was closed with Unifast (multipurpose room temperature polymerization resin, product name, manufactured by GC Corporation). Wash with 2 mL of 5% sodium hypochlorite and 5 mL saline, and apply smear clean (3% EDTA aqueous solution, product name, manufactured by Nippon Dental Co., Ltd.) into the root canal for 2 minutes at 4 ° C. It was stored in physiological saline. The inside of the root canal was dried with a brooch cotton plug. As a drug, final concentration 0.006% (w / v) antibacterial nanoparticles, 0.2% chlorhexidine gluconate, 0.02% benzalkonium chloride, 0.12% cetylpyridinium chloride, final concentration 35 μg / mL doxycycline hydrochloride Using salt hydrate, it was mixed with minus or plus nanobubble water, or used alone as a drug (diluted with Otsuka distilled water). Further, to detect drug penetration, autofluorescent tetracycline was mixed at a final concentration of 5 mg / mL. The drug solution was transported into the root canal with a pipette and applied for 5 minutes to allow the drug to penetrate into the root canal dentinal tubule. After washing with physiological saline, the chemical solution was removed with a brooch cotton plug, and the inside of the root canal was dried. The teeth are placed on a metal base so that the pulp cavities are parallel. Utility wax (product name, manufactured by Cabo Dental Systems Japan Co., Ltd.), Unifast III (ultrafast-hardening room temperature polymerization resin, product name, manufactured by GC Corporation). It was fixed at. When Unifast III was cured, a section specimen having a thickness of about 300 μm was prepared by Seege Microtome (product name, manufactured by Microsystems, Inc. next summer) and observed with a stereoscopic fluorescence microscope.

5A-5F are fluorescence stereomicrographs in which a fluorescent agent (tetracycline) is infiltrated from the root canal wall (RC) in the pig tooth root to the deep part in the dentinal canal. In addition, Table 2 shows the penetrance into the deep part of the ivory canaliculus in three stages (++, +,-). As shown in FIGS. 5 and 3, with respect to antibacterial nanoparticles, both minus nanobubble water and plus nanobubble water showed significant promotion of penetration into the deep dentinal tubules as compared with distilled water (B). In benzalkonium chloride, there was no difference depending on whether the nanobubbles were charged positively or negatively (D), but chlorhexidine gluconate (C), cetylpyridinium chloride (E) and doxycycline hydrochloride hydrate (F). ), The plus nanobubble water showed a significant promotion of permeation as compared with the minus nanobubble water. From these results, it can be said that the effect of promoting the penetration of the antibacterial agent into the deep part of the dentinal tubule is superior to that of the plus nanobubble water than that of the minus nanobubble water.

[Example 4]
(Removal of porcine root canal dentin smear layer)
A pseudo root canal of a pig tooth that has been pre-cut with a Disc (polishing disc) to make it easier to break is formed by expanding the root canal to # 70 with a K file (product name, manufactured by Manny Co., Ltd.), and 5% hypochlorous acid. Alternate washing was performed with 2 mL of sodium acid acid and 2 mL of 3% hydrogen peroxide solution. It was further washed with 5 mL saline and stored in saline at 4 ° C. Using 0.2% chlorhexidine gluconate and 0.12% cetylpyridinium chloride as chemicals, mix with minus or plus nanobubble water or distilled water from Otsuka, and apply 2 mL of 9 types of smear layer remover for 5 minutes. The smear layer was washed. After washing with physiological saline, it was divided in half with tooth-extracted citrus, fixed with 2% glutaraldehyde for 12 hours, dehydrated with 30, 50, 70, 90, 100% ethanol, and then vapor-deposited with platinum 10 kV. .. The vapor deposition was carried out by conductive film deposition (sputter coating) using a magnetron sputtering apparatus (product name: MSP-20-UM, manufactured by Vacuum Device Co., Ltd.). Then, each specimen was observed at the central part of the root canal with a scanning electron microscope (product name: VE9800, manufactured by KEYENCE CORPORATION).

When the number of ivory tubules after washing was measured, the smear layer could hardly be removed with distilled water, but when used alone (without chemicals), plus nanobubble water significantly promoted smear layer removal than minus nanobubble water. ( ^## P <0.01) (Fig. 6A, Fig. 7). On the other hand, 0.2% chlorhexidine gluconate and 0.12% cetylpyridinium chloride had the effect of significantly promoting the removal of the smear layer when used in combination with plus nanobubble water than when used in combination with minus nanobubble water. ( ^# P <0.05) (FIGS. 6B, 6C, 7).

The present invention can be used for caries treatment, demyelination / infected root canal treatment, periodontal disease treatment, and oral care.

Claims (9)

Contains antibacterial agents and nanobubbles,
The nanobubble is a composition for dentistry and oral cavity, characterized in that the surface thereof is a positively charged plus nanobubble. The dental or oral composition according to claim 1, wherein the zeta potential of the nanobubbles is positive. The concentration of the plus nanobubbles is 1 × 10 ⁶ to 1 × 10 ⁹ cells / mL.
The average bubble diameter of the plus nanobubbles is 10 to 300 nm.
The dental or oral composition according to claim 1 or 2. The antibacterial agent is an antibacterial nanoparticle, an antibiotic, an antibacterial agent, an antifungal agent, an antiviral agent, a disinfectant, a root canal enlarger, an anti-inflammatory agent, a physiologically active substance that promotes wound healing and tissue regeneration, and a stem cell-derived secretome. The dental or oral composition according to claim 1 or 2, which comprises any one or more of the groups consisting of exosomes and miRNAs. The dental or oral composition according to claim 1 or 2, wherein the antibacterial agent is a cationic antibacterial agent. A composition for treating bacterial infection in the root canal of a tooth, which comprises the composition for dental and oral cavity according to any one of claims 1 to 5. A composition for treating bacterial infection in the periodontal tissue of a tooth, which comprises the composition for dental and oral cavity according to any one of claims 1 to 5. A composition for treating bacterial infection of dental caries, which comprises the composition for dental and oral cavity according to any one of claims 1 to 5. A composition for treating bacterial infection of tongue coating, which comprises the composition for dental and oral cavity according to any one of claims 1 to 5.

Images