JP7101365B2 - Pulp capping agent - Google Patents

Description

The present invention relates to a pulp capping agent for dental pulp capable of inducing odontoblast differentiation from dental pulp and further promoting dentin calcification, which is the final differentiation.

The tooth has a structure in which the outermost surface is covered with enamel composed of hydroxyapatite, the dentin is inside, and the cavity in the dentin is the pulp consisting of connective tissue including blood vessels and nerves. .. There is a single layer of odontoblasts on the inner surface of the dentin in contact with the pulp, and it is known that the dentin is thickened by physiological or pathological stimuli.

In the dental preservation treatment of cases where caries has progressed to deep dentin, after removing the dentin softened by the infection of the causative bacteria of caries, an indirect pulp covering method is applied to protect the pulp, and the pulp is sterile. If the pulp is partially exposed, the pulp covering method is applied directly to the exposed pulp surface, or if the infected dentin is expected to be removed, the infected dentin is intentionally left and the pulp covering material is left. Is applied to sterilize and remineralize the infected dentin and treat it with a temporary indirect pulp covering method that expects the formation of repaired dentin.

Since conservative treatment is difficult when the pulp becomes inflamed, it is a condition that there is no spontaneous pain due to inflammation in the direct pulp capping method or the temporary indirect pulp capping method. When the pulp becomes irreversible, conservative treatment is abandoned, and treatment is performed to block the pulp cavity with a root canal filling material consisting of pulp removal and gutter percha. Root canal-filled teeth lose their nutrient supply to dentin and are prone to tooth fracture due to lack of strength. In recent years, it has been considered important to preserve the pulp as much as possible and leave healthy teeth.

Currently, calcium hydroxide preparations, carboxylate cements containing tannin / fluoride mixture, and mineral trioxide aggregate (MTA) are mainly used as pulp capping agents. Calcium hydroxide preparations are thought to promote sterilization and dentin calcification by stimulation with strong alkaline calcium hydroxide. Further, the carboxylate cement containing a tannin / fluoride mixture is expected to have a bactericidal effect due to tannin and an dentin calcification promoting effect due to fluoride (Non-Patent Document 1). Furthermore, mineral trioxide agrigate (MTA) contains dicalcium silicate, tricalcium silicate, tricalcium aluminate, gypsum, and bismuth oxide, and has a bactericidal action by strong alkali and a hard tissue forming action by calcium release action. Although its dental pulp tissue reaction has many similarities to calcium hydroxide preparations, it is said to be superior to calcium hydroxide preparations. That is, the dental pulp in contact with MTA is degenerated, and the cells proliferated and migrated from the surroundings differentiate into odontoblasts to form hard tissue (Non-Patent Document 2), which is common to these pulp capping agents. Due to its strong irritation caused by strong alkali, it cannot be used for patients with hypersensitivity such as rash and dermatitis.

Examples of the component that promotes the formation of ivory include bovine blood extract (Patent Document 1), polysaccharides such as glucosaminoglycan (Patent Document 2), BMP-2 (bone morphogenetic protein-2), and the like. Growth factors such as bone morphogenetic factor (Patent Document 3), HMG-CoA reductase inhibitor (Patent Document 4), and TGF-β1 (Patent Document 5) have been reported.

By the way, geranylgeraniol is known as an aromatic substance in essential oils used as a fragrance, and is known to exhibit an anti-inflammatory effect (Non-Patent Document 3). Patent Document 6 discloses an oral composition containing polyphosphoric acid, geranylgeraniol, and the like.

JP-A-2002-3633084 Japanese Unexamined Patent Publication No. 06-256132 Japanese Unexamined Patent Publication No. 06-340555 International Publication No. 2008/120720 Pamphlet International Publication No. 2007/013430 Pamphlet Japanese Unexamined Patent Publication No. 02-59512

Japanese Society of Conservation of Dentistry AIPC (Non-Invasive Pulp Cover) Guidelines Kunihiko Yoshiba et al., Journal of Niigata Dental Society, 39,181-182,2009 Marcuszi A et al., Int Immunopharmacol, 10 (6): 639-642, 2010.

The dentin formation promoting action of the pulp capping material containing the above-mentioned calcium hydroxide preparation, carboxylate cement containing tannin / fluoride mixture, or mineral trioxide agrigate (MTA) is the strong alkali contained in the pulp capping material. Inorganic stimulation causes pulp degeneration, and the restoration mechanism causes dentin calcification, but the restoration takes time and the prognosis is uncertain, as well as patients with dermatitis and hypersensitivity. There are restrictions such as not being able to use it. In addition, bovine blood and polysaccharides, which are said to promote dentin differentiation, may be contaminated with unknown sources of infection and contaminants. Bone formation factors and growth factors are expensive because they must be produced by gene recombination technology and further purified. Statins, which are HMG-CoA reductase inhibitors, remain problematic with side effects such as rhabdomyolysis.
In the oral composition described in Patent Document 6, geranylgeraniol is merely blended as a component for stabilizing polyphosphoric acid, which has an effect of suppressing the formation of tartar, and the oral composition. Is only intended for use as dentifrices, mouthwashes, troches, chewing gum, etc.
In view of such a situation, it is an object of the present invention to provide a pulp capping agent for dental pulp capable of inducing differentiation of odontoblasts from dental pulp and further promoting dentin calcification, which is the final differentiation.

The present inventor has conducted extensive research to solve the above problems. As a result, it was found that the geranylgeraniol derivative known as an aromatic substance in the essential oil used as a fragrance promotes the differentiation of odontoblasts from the dental pulp and promotes the final differentiation, dentin calcification. , The present invention has been completed.
Hereinafter, the present invention will be shown.

[1] A pulp capping agent comprising a geranylgeraniol derivative as an active ingredient.
[2] The above-mentioned geranylgeraniol derivative is one or more compounds selected from the essential group from geranylgeraniol, prounotor, abietic acid, carotene, geranylgeranylpyrophosphate, farnesylpyrophosphate, geranylpyrophosphate, and geraniol. The myelinated agent according to [1].
[3] The pulp capping agent according to the above [1] or [2], wherein the geranylgeraniol derivative is contained in biodegradable microcapsules.
[4] The spinal cord according to any one of the above [1] to [3], wherein the biodegradable microcapsules are essential from polyglycolic acid, polylactic acid, or a lactic acid-glycolic acid copolymer. Agent.
[5] The pulp capping agent according to the above [3] or [4], wherein the biodegradable microcapsules are dispersed in an ointment base.

The gelanyl gelaniol derivative, which is the active ingredient of the pulp capping agent according to the present invention, does not cause dental pulp degeneration due to strong alkali and promote dentin calcification unlike the conventional major pulp capping agents such as calcium hydroxide preparations. Therefore, it is safe and can be used for patients with dermatitis and irritability. In addition, geranylgeraniol derivatives are so-called terpenes and have a relatively simple chemical structure unlike proteins, so it is possible to isolate high-purity derivatives from nature, or high-purity derivatives. It can be said that it is safe and inexpensive in that it can be chemically synthesized. Further, the geranylgeraniol derivative is excellent in biocompatibility because it is derived from a living body or can be synthesized from a compound derived from a living body. In addition, the present inventor has experimentally found that a geranylgeraniol derivative induces differentiation from dental pulp into odontoblasts and exhibits an excellent dentin calcification effect. Therefore, the pulp capping agent according to the present invention is extremely useful as a pulp capping agent for dental pulp used for the treatment of dental caries.

FIG. 1 is a photograph of isolated dental pulp cells cultured in undifferentiated medium or differentiation medium in the presence or absence of geranyl gelaniol, and the calcification of odontoblasts visualized by Arizarin Red S staining and Voncossa staining. be. FIG. 2 is a graph showing the amount of geranylgeraniol released over time from the sustained release microcapsules according to the present invention.

The pulp capping agent according to the present invention contains a geranylgeraniol derivative as an active ingredient.
In general, geranylgeraniol derivatives include isoprene units derived from mevalonic acid as the basic skeleton, and the final products such as prounotor, avietinic acid, and α-, β- or γ-carotene, as well as these final products. Includes all biosynthetic intermediates geranylgeraniol, geranylgeranylpyrophosphate, and precursors thereof such as farnesylpyrophosphate, geranylpyrophosphate, geraniol and the like. The chemical structural formulas of these compounds are shown below.

Proutol is used as a gastritis drug and is thought to be produced by the introduction of a hydroxyl group into geranylgeraniol or geranylgeraniolpyrrophosphate, but geranylgeraniol itself is known to have anti-inflammatory effects.

Geranyl geraniol is mainly synthesized by a chemical synthesis method, but when chemically synthesized, one or more double bonds having the same carbon skeleton are synthesized as a mixture with a cis form, so that it is industrially synthesized. Various methods have been disclosed for the production of geranyl gelaniol in which all important double bonds are trans (see JP-A-9-238692, JP-A-2005-137287, etc.). On the other hand, geranylgeraniol is extracted from the seeds of annatto (Bixaceae, Achiote), a plant native to Central to South Africa. Annatto pigments, which have been extracted as carotenoid pigments for a long time, are used in various processed foods as food additives because they are acidic and yellow and neutral red, but they are also used in cosmetic lipsticks. There is. Furthermore, recently, a method for recovering geranylgeraniol from the residue of the extract from which the annatto pigment has been removed has been shown (see US Pat. No. 6,350,453). The geranylgeraniol used in the present invention may be chemically synthesized according to a known method or may be extracted. The other geranylgeraniol derivatives may be chemically synthesized, may be of natural origin, or may be synthesized from other geranylgeraniol derivatives as raw materials.

Geranylgeraniol is chemically synthesized and used as a raw material for vitamin E, vitamin K2, and the gastrointestinal drug pranolol. Chemical Industry Co., Ltd.) and the trade name "geranylgeraniol-50" (manufactured by Mitsubishi Chemical Foods Co., Ltd.) are used in cosmetics and health foods.

As the pulp capping agent according to the present invention, a sustained-release pulp capping agent in which a geranylgeraniol derivative is contained in biodegradable microcapsules and the geranylgeraniol derivative is gradually released is preferable. While the conventional pulp capping agent stimulates the dental pulp with strong alkali to promote calcification, the sustained release pulp capping agent according to the present invention releases calcification by sustained release of the gelanyl gelaniol derivative which is an active ingredient. Sustainable promotion of calcification.

The biodegradable polymer constituting the biodegradable microcapsules is not particularly limited as long as it can be decomposed in vivo to release a geranylgeraniol derivative, and is not particularly limited, and is, for example, α-hydroxycarboxylic acid, hydroxydicarboxylic acid, and hydroxytricarboxylic acid. A polycondensation homopolymer or copolymer of at least one hydroxycarboxylic acid selected from the group consisting of essential acids is preferred, and polymers with repeating units derived from α-hydroxycarboxylic acid are more preferred. Further, examples of the biodegradable polymer include polyglycolic acid, polylactic acid, and a lactic acid-glycolic acid copolymer, and one or more of these may be used, but more preferably, lactic acid-glycol. It is an acid copolymer.

The polyglycolic acid, polylactic acid, and lactic acid-glucoric acid copolymers used in the present disclosure may be D-forms, L-forms, or mixtures thereof, but racemates are preferred.

The weight average molecular weight of polyglycolic acid and polylactic acid is preferably 10,000 or more and 28,000 or less, and the weight average molecular weight of the lactic acid-glycolic acid copolymer is preferably 4,000 or more and 116,000 or less. , 24,000 or more and 38,000 or less are more preferable.

The copolymerization ratio (lactic acid / glycolic acid (mol%)) of the lactic acid-glycolic acid copolymer is preferably 15/85 to 50/50, more preferably 25/75 to 35/65.

The terminal group of the polyglycolic acid, polylactic acid, and lactic acid-glycolic acid copolymer may be either an alkyl ester or a carboxylic acid, and an alkyl ester is preferable when long-term sustained-release property is required, but sustained-release control is performed. You can choose according to your purpose.

The particle size of the biodegradable microcapsules may be appropriately adjusted, but for example, it may be large enough to enclose a geranylgeraniol derivative, and may be dispersible in an ointment base or may be passed through after being liquid-dispersed as it is. Considering the needle property, it is preferably 1 μm or more and 100 μm or less, but is not limited thereto.

The sustained release pulp capping agent in the present invention
The step of preparing a solution containing at least a geranylgeraniol derivative, a biodegradable polymer, and a lipophilic solvent,
A step of preparing an O / W type microemulsion by dispersing the above solution in an aqueous solvent, and
It can be produced by a method including a step of distilling off the lipophilic solvent from the O / W type microemulsion.

The lipophilic solvent that dissolves the geranyl gelaniol derivative and the biodegradable polymer exhibits moderate solubility in the geranyl gelaniol derivative and the biodegradable polymer, and is insoluble or sparingly soluble in water, and is liquid at room temperature. There are no particular restrictions as long as they are present. Here, "insoluble or sparingly soluble in water" means that 1000 mL or more of water is required to dissolve 1 g or 1 mL of lipophilic solvent at 25 ± 5 ° C. Examples of the lipophilic solvent include vegetable oils such as palm oil, rapeseed oil, olive oil, and sesame oil; tri-, di-, mono-fatty acid glycerides such as stearic acid, palmitin glyceride, and laurin glyceride; n-heptane, n-octane, and fluid. Aliphatic hydrocarbon solvents such as paraffin; aromatic hydrocarbon solvents such as toluene, monochlorobenzene and dichlorobenzene; halogenated hydrocarbon solvents such as dichloromethane, monofluoromethane, difluoromethane and perfluorohexane; ethyl acetate and butyl acetate and the like. Examples of the fatty acid ester solvent of. Further, a mixed solvent of two or more of these may be used, or a hydrophilic solvent such as methanol or ethanol may be used in combination if necessary for dissolving the geranylgeraniol derivative and the biodegradable polymer. ..

Metal salts and / or metal oxides may be added to the above solutions. By adding such a metal salt / metal oxide, the encapsulation rate of the geranyl gelaniol derivative in the biodegradable polymer may be increased, or the differentiation of odontoblasts and bone formation may be promoted. In addition, it can be expected that calcification will be further promoted by lithium ions and the like. Examples of the metal constituting the metal salt / metal oxide include alkali metals such as lithium, sodium and potassium; second-general elements such as calcium and magnesium; base metals such as zinc; transition metals such as titanium and zirconium. Can be mentioned. Examples of the metal salt include inorganic acid salts such as hydrochloride, sulfate, nitrate and thiocyanate; aliphatic carboxylates such as acetate; aromatic carboxylates such as benzoate and the like. Be done.

The concentration of each component in the above solution may be appropriately adjusted, and for example, the concentration of the biodegradable polymer can be 30% by mass or more and 80% by mass or less. The ratio of the geranylgeraniol derivative to the biodegradable polymer can be 1% by mass or more and 30% by mass or less, preferably 2% by mass or more and 30% by mass or less. The concentration of the metal salt / metal oxide in the above solution is preferably 0.001% by mass or more and 1% by mass or less.

Next, the above solution is dispersed in an aqueous solvent to prepare an O / W type microemulsion. The aqueous solvent refers to water and a mixed solvent of water and a water-miscible organic solvent. The water-miscible organic solvent means a hydrophilic organic solvent that can be miscible with water without limitation, and for example, a lower alcohol solvent such as methanol, ethanol, and isopropanol; and a polyhydric alcohol solvent such as ethylene glycol, propylene glycol, and glycerin. Can be mentioned. The ratio of the water-miscible organic solvent in the mixed solvent may be appropriately adjusted, but is preferably 40% by mass or less, more preferably 20% by mass or less or 10% by mass or less, still more preferably 5% by mass or less.

An emulsion stabilizer such as a water-soluble polymer or a surfactant may be used to stabilize the microemulsion. Examples of the water-soluble polymer include polyethylene glycol, polypropylene glycol, polyvinyl alcohol and the like. As the surfactant, a nonionic surfactant such as sorbitan aliphatic ester can be used. The amount of the emulsion stabilizer used may be appropriately adjusted, and may be, for example, 0.01% by mass or more and 1% by mass or less with respect to the aqueous solvent.

The ratio of the above solution to the aqueous solvent may be appropriately adjusted. For example, the ratio of the solution to the total of the solution and the aqueous solvent can be 0.1% by mass or more and 20% by mass or less, preferably 0.5% by mass or more and 10% by mass or less.

As a method for preparing the O / W type microemulsion, a conventional method can be used. For example, after mixing the above solution and an aqueous solvent, the mixture may be stirred and mixed using a propeller-type or turbine-type stirrer, a homomixer, or the like.

Next, by distilling off the lipophilic solvent from the O / W type microemulsion, biodegradable microcapsules containing a geranylgeraniol derivative can be prepared. For distilling off the lipophilic solvent, a conventional method such as depressurization and / or heating may be used. When a lipophilic solvent having a boiling point lower than that of the aqueous solvent is used, the lipophilic solvent can be selectively distilled off.

After distilling off the lipophilic solvent, it is preferable to carry out a normal post-treatment. For example, when a lipophilic solvent having a boiling point lower than that of an aqueous solvent is used, a dispersion of biodegradable microcapsules can be obtained by removing the lipophilic solvent. The biodegradable microcapsules may be separated from the aqueous solvent by filtration, centrifugation or the like, washed with the aqueous solvent, and finally dried. Further, a part or all of the aqueous solvent may be distilled off together with the lipophilic solvent. Even in that case, the biodegradable microcapsules can be washed and isolated in the same manner.

The dosage form of the pulp capping agent according to the present invention is not particularly limited, and a dosage form suitable for the treatment of dental caries, such as an ointment or a liquid, can be used. In addition, the pulp capping agent of the present invention can be expected to be a more effective therapeutic agent by adding other medicinal ingredients such as a bactericidal agent, an antibiotic, and an anti-inflammatory agent, if necessary. The spinal cord covering agent of the present invention includes a base, an excipient, a colorant, a lubricant, a flavoring agent, an emulsifier, a thickener, a wetting agent, a stabilizer, a preservative, and a solvent, depending on the dosage form and the like. , Dissolving aids, suspending agents, antioxidants, additives, buffers, pH regulators, sweeteners, fragrances and the like can be blended. For example, ointment bases include hydrophobic bases such as white petrolatum, yellow petrolatum, liquid paraffin, plastibase ^(R) , silicon, vegetable oil, pork fat, and honeydew, and hydrophilic petrolatum, refined lanolin, macrogol ^(R) , etc. A hydrophilic base can be mentioned.

The ratio of the geranylgeraniol derivative, which is the active ingredient in the pulp capping agent according to the present invention, is not particularly limited and may be appropriately adjusted, but may be, for example, about 20% by mass or more and 80% by mass or less.

More specifically, for example, a sustained release spinal cord containing a geranyl gelaniol derivative in biodegradable microcapsules is used as a base for the existing spinaching agent TMR-MTA cement (calcium silicate, It can be dispersed and mixed with calcium aluminate, zirconium oxide, silicon dioxide, etc.) and administered to the affected area. Further, after directly administering the pulp capping agent according to the present invention such as a liquid agent or an ointment to the affected area, it is coated with TMR-MTA cement or a photopolymerizable composite resin for dental filling composed of an organic resin matrix and an inorganic filler. It can also be protected.

The pulp covering agent of the present invention promotes the induction of differentiation of odontoblasts from the dental pulp by releasing the geranyl gelaniol derivative from the exposed pulp surface or the infected dentin into the dental pulp, promotes dentin calcification, and geranyl gelaniol. The anti-inflammatory action of the derivative relieves inflammation of the affected area, forms repaired dentin, and can prevent pulp inflammation and dentin damage due to pressurization of the repaired material.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples as well as the present invention, and appropriate modifications are made to the extent that it can meet the purposes of the preceding and the following. Of course, it is possible to carry out, and all of them are included in the technical scope of the present invention.

Example 1: Preparation of microcapsules Using a lactic acid-glycolic acid copolymer as a biodegradable polymer, geranyl gelaniol was microencapsulated via an O / W type emulsion.
Specifically, 2.0 g of a lactic acid-glycolic acid copolymer (copolymerization ratio: 50/50, weight average molecular weight: 24,000 to 38,000), 1 mg of zinc oxide, and 25 mg of geranyl geraniol were added to 4 mL of methanol. A drug-biodegradable polymer solution (hereinafter referred to as "O phase") was prepared by dissolving in a mixed solvent of 1 mL of methanol.
Polyvinyl alcohol (manufactured by "Poval" Kuraray Trading Co., Ltd.) was dissolved in water as an emulsifier to prepare a 0.1% by mass aqueous solution (hereinafter referred to as "W phase").
After adding the above W phase 445 mL to the glass beaker and then the above O phase 5 mL, emulsify with a homomixer (“MODEL HM-300” manufactured by HSIANGTAI) at 6,000 rpm for 1 minute, and mix (hereinafter referred to as “mixture”). "O / W type emulsion") was obtained.
The O / W type emulsion was transferred to a 1 L eggplant type flask, and the organic solvent was distilled off in water at 25 ° C. for 2 hours using a rotary evaporator (“N-1N type” manufactured by Tokyo University of Science). The mixture was then placed in a centrifuge tube and centrifuged at 3,000 rpm for 10 minutes to remove the supernatant. Purified water was added to the obtained sediment, and the mixture was centrifuged again at 3,000 rpm for 10 minutes to remove the supernatant. Then, a small amount of purified water was added to the sediment and the suspension was well suspended. This suspension was suction-filtered using Buchnaroth (“3G No. 1” manufactured by Fuji Science Industry Co., Ltd., pore diameter: 100 to 120 μm).
The obtained filtrate was transferred to a eggplant-shaped flask, pre-frozen at −40 ° C., then heated at 10 Pa for 12 hours, then heated to −10 ° C., vacuum freeze-dried for 24 hours, and further heated to 25 ° C. Vacuum drying was performed for 48 hours to obtain sustained release microcapsules of geranylgeraniol.

Example 2: Preparation of microcapsules 2.0 g of lactic acid-glycolic acid copolymer (copolymerization ratio: 50/50, weight average molecular weight 7,000 to 17,000) and 25 mg of geranyl geraniol in 4 mL of dichloromethane and 1 mL of methanol. Geranylgeraniol sustained-release microcapsules were obtained in the same manner as in Example 1 above except that a drug-biodegradable polymer solution (Phase O) was prepared by dissolving in a mixed solvent.

Example 3: Preparation of microcapsules 2.0 g of lactic acid-glycolic acid copolymer (copolymerization ratio: 65/35, weight average molecular weight: 24,000 to 38,000), 1 mg of zinc oxide, and 25 mg of geranyl geraniol. Geranylgeraniol sustained-release microcapsules were obtained in the same manner as in Example 1 above except that a drug-biodegradable polymer solution (Phase O) was prepared by dissolving in a mixed solvent of 4 mL of dichloromethane and 1 mL of methanol.

Example 4: Preparation of microcapsules 2.0 g of lactic acid-glycolic acid copolymer (copolymerization ratio: 75/25, weight average molecular weight: 4,000 to 15,000), 1 mg of zinc oxide, and 25 mg of geranyl geraniol. Geranylgeraniol sustained-release microcapsules were obtained in the same manner as in Example 1 above except that a drug-biodegradable polymer solution (Phase O) was prepared by dissolving in a mixed solvent of 4 mL of dichloromethane and 1 mL of methanol.

Test Example 1: Evaluation of calcification effect (1) Isolation and culture of dental pulp stem cells For isolation and culture of dental pulp cells from mouse cut teeth, a method such as Akmal MRN (Journal of Biological Sciences, 14 (4): The modification method of 327-331, 2014 doi: 10.2393 / jbs. 2014.327.331) was used. Specifically, phosphoric acid containing type 1 collagenase (2 mg / mL, manufactured by Wako Pure Chemical Industries, Ltd.) and dispase II (4 mg / mL, manufactured by Adia) after removing the cut tooth pulp from the mandible of a mouse and chopping it into small pieces. Digested in buffer (PBS) at 37 ° C. for 30 minutes with shaking. A micropipette tip with a volume of 1000 μL and then a micropipette with a volume of 200 μL were used to loosen the digest while pumping. The obtained cell suspension was filtered with a cell strainer (manufactured by Falcon) having a diameter of 40 μm to remove undigested tissue, and the filtrate containing cells was centrifuged at 1,000 rpm for 5 minutes. The obtained cell pellet is suspended in α minimum essential medium containing 20% fetal bovine serum (“α-MEM” manufactured by Wako Pure Chemical Industries, Ltd.), seeded in a culture dish (manufactured by Falcon), and then 5% CO ₂ Cultured below. The medium was changed every 3 days.

(2) Confirmation of the effect of geranyl geraniol on odontoblasts Cells that reached confluence by culture were treated with 10% activated charcoal-treated fetal bovine serum (HyClone), 50 mM ascorbic acid (Sigma-Aldrich), and 10 mM β. -The cells were differentiated into odontoblasts by exchanging with α-MEM differentiation-inducing medium containing glycerophosphate (manufactured by Sigma-Aldrich). Medium exchange was performed every 3 days.
Simultaneously with the change of medium to the differentiation-inducing medium, geranylgeraniol (manufactured by Sigma-Aldrich) dissolved in dimethyl sulfoxide (DMSO) was added to a concentration of 10 μM or 20 μM. As a control, a differentiation-inducing medium supplemented with the same amount of DMSO or an undifferentiated medium of α-MEM containing serum was used. Alizarin Red S staining and von Kossa staining were performed on the 15th day after the medium was replaced with the differentiation-inducing medium, and calcification was evaluated.
Specifically, after inducing differentiation, the cells were washed with cooled PBS and fixed with cooled 70% ethanol for several minutes. After air-drying, the cells were stained with 40 mM alizarin red S (manufactured by Cosmo Bio Co., Ltd., pH 4.2) at room temperature for 10 minutes, washed with deionized water, and then examined under a microscope. In addition, cells were fixed at room temperature for 30 minutes with PBS (manufactured by Wako Pure Chemical Industries, Ltd.) containing 10% formalin for von Kossa staining. After washing with deionized water, the color was developed under a fluorescent lamp for 30 minutes with a 5% aqueous silver nitrate solution. 5% sodium thiosulfate was added and the reaction was stopped. After washing with deionized water, the microscope was examined. The results are shown in FIG.
As shown in FIG. 1, dental pulp cells cultured for 15 days in an undifferentiated medium were not stained with alizarin red staining and von Kossa staining, which are indicators of calcification, probably because they were undifferentiated. On the other hand, when the medium was replaced with a differentiation medium, positive stained images by alizarin red staining and von Kossa staining appeared on the 15th day of culture, indicating that the cells differentiated into odontoblasts. Furthermore, when cultured in a differentiation medium containing geranylgeraniol, strongly positive images in alizarin red staining and von Kossa staining depending on the concentration of geranylgeraniol added appeared. These results demonstrate that the addition of geranylgeraniol promotes the differentiation and calcification of dental pulp stem cells into odontoblasts.

Test Example 2: Release test of geranyl gelaniol from sustained-release microcapsules 0.5 g of sustained-release microcapsules prepared in Example 3 above are mixed with 1 mL of 0.1 M phosphate buffer having a pH of 7.4 and suspended. Prepare 6 samples added to a dialysis tube (“RC Membrane Pore 3” manufactured by Ieda Trading Co., Ltd., MWCO: 3,500) that was prepared and swollen with 0.1 M phosphate buffer (pH 7.4) in advance. Then, it was immersed in 100 mL of 0.1 M phosphate buffer (pH 7.4) and dialyzed at room temperature. The dialysis tube was taken out on the 1st, 3rd, 7th, 14th, 21st, and 28th days and used as a test sample.
The test sample was placed in an Eppendorf chamber, centrifuged at 12,000 rpm for 1 minute, the supernatant was carefully removed, and 4 mL of n-hexane was added to the precipitate to prepare a sample. In addition, a standard solution in which 10 mg of geranylgeraniol was dissolved in 20 g of n-hexane was prepared. The quantification of geranylgeraniol was performed by HPLC according to a known method (EK Silva et al., Food Research International, 78: 159-168, 2015). That is, a C18 column heated to 40 ° C. (manufactured by KINETEX, 100 mm × 4.6 mm × 2.6 μm) and methanol / 50 mM ammonium acetate solution = 90/10 (V / V) were used as the mobile phase, and the flow rate was 1 mL. Geranylgeraniol was detected at / min and an absorbance of 210 nm. The concentration of the prepared geranylgeraniol standard solution was adjusted to various concentrations using hexane, 10 μL of each was collected, and HPLC-PDA (“Alliance E2695” manufactured by Waters) was passed through a nylon film filter (0.45 μm). It was quantified using and a calibration curve was prepared. From the measured values of the sample, the sustained release amount of geranylgeraniol was determined using the calibration curve and the following formula. The results are shown in FIG.
Sustained release amount (% by mass) = [(content in test sample-content in precipitate) / content in test sample] x 100
As shown in FIG. 2, it was demonstrated that the Geranylgeraniol sustained release microcapsules according to the present invention can continuously release Geranylgeraniol.

Claims (5)

A pulp capping agent characterized by containing geranylgeraniol as an active ingredient. The pulp capping agent according to claim 1, wherein the geranylgeraniol is contained in biodegradable microcapsules. The spinal cord covering agent according to claim 1 or 2 , wherein the biodegradable microcapsules are essential from polyglycolic acid, polylactic acid, or a lactic acid-glycolic acid copolymer. The pulp capping agent according to claim 2 or 3 , wherein the biodegradable microcapsules are dispersed in an ointment base. A method for producing sustained release pulp capping agents,
The step of preparing a solution containing at least geranylgeraniol, a biodegradable polymer, and a lipophilic solvent,
A step of preparing an O / W type microemulsion by dispersing the above solution in an aqueous solvent, and
A method comprising a step of distilling off the lipophilic solvent from the O / W type microemulsion.

Images