Dental composition, method for producing dental composition, and method for culturing dentin

Abstract

A composition 4 contains a ground product 3 obtained by grinding human teeth, and provides appropriate scaffolds for human dental pulp cells since dentine and dentinal tubules are present in a cross-section of a tooth. Further, it possible to induce odontoblasts from human dental pulp cells and cause regeneration / reparation of dentine, without bringing unease to a patient about medicinal agents since human teeth is used. Therefore, by using a dental composition containing a ground product of human teeth, it is possible to cause reparation / regeneration of dental pulp tissues, and makes it easy to assure the safety of materials used in a regenerative therapy for dental pulp.

Description

[Technical field]

[0001] The present invention relates to a dental composition and a method for culturing dentin, which are used for treating dental caries, pulpectomy, infected root canal treatment, etc. [Background technology]

[0002] The structure of a tooth is made up of layers, from the outside in, which are enamel, dentin, and dental pulp. Dentin is one of the hard tissues that make up teeth, and is composed of about 70% inorganic matter and about 30% organic matter. Dentin is characterized by being softer than enamel, which is composed mostly of inorganic matter. With soft dentin layered underneath the hard enamel, the dentin acts like a cushion, giving the tooth as a whole a strong structure that is resistant to cracking even when subjected to impact.

[0003] However, as tooth decay progresses and dentin is eroded, the dental pulp beneath the dentin surface gradually moves closer to the tooth surface, becoming exposed and connected to the oral cavity through dentinal tubules. Because the dental pulp is nerve tissue, when the pulp moves closer to the tooth surface, subjective symptoms of tooth decay such as tooth sensitivity and pain are felt. When tooth decay progresses and symptoms such as pulpitis appear, a pulpectomy is generally performed to remove the inflamed pulp.

[0004] The dental pulp has the function of notifying tooth decay by pain sensation and the cushioning effect of the dental pulp tissue itself, which prevents fractures, and protects the tooth by metabolic action by blood vessels and nerves, anti-inflammatory and anti-infection effects, etc. Therefore, pulp extraction increases the risk of tooth loss due to fractures, spread of infection to the apical periodontal tissue, or worsening of inflammation. Treatments for such situations in teeth that have had their pulp extracted include dentures and implant treatments. However, these treatments have the problem of causing a decrease in esthetics and occlusion. Therefore, a new treatment method, dental pulp regeneration therapy, which restores dental pulp tissue to its original state after pulp extraction, has been proposed. This treatment involves harvesting dental pulp tissue from unused teeth such as wisdom teeth, culturing the dental pulp stem cells present in the tissue, and transplanting these and drugs into the teeth that have had their pulp extracted to regenerate dental pulp tissue. However, while this treatment regenerates the entire pulp tissue and adds dentin to the dentin walls of the root canal, it can only induce a small amount of dentin regeneration in the crown, and the current situation is that this is reinforced with caps and fillings such as cement, resin, and metal.

[0005] Regarding the approach to regenerating dentin tissue, a chemical method has been proposed in which a drug is added to promote differentiation induction of dental pulp cells. For example, Patent Document 1 describes a method of regenerating dentin in which dental pulp cells are differentiated into odontoblasts using a composite material in which non-collagenous phosphorylated proteins such as phosphorin are crosslinked to collagen as a scaffold. Patent Document 2 describes a dentin formation promoter containing an HGM-CoA reductase inhibitor as an active ingredient. Patent Document 3 describes a method for producing differentiation-induced odontoblasts, which includes culturing isolated human dental pulp cells three-dimensionally while adding 1,25 (dihydroxy) vitamin D3 to a predetermined cell density. However, since odontoblasts induced to differentiate in a test tube as in these chemical approaches are terminally differentiated, they cannot adhere even when transplanted into a living body, and it is considered difficult to use them for dentin regeneration.

[0006] In contrast to the chemical approach, which is a scheme in which odontoblasts are produced and then transplanted, a physical approach using artificial inorganic fired materials has been proposed, in which dental pulp cells in a pulp-excised tooth are differentiated into odontoblasts. For example, Patent Document 4 describes a method for regenerating dentin using a ceramic porous carrier. However, the ceramic porous carrier described in the same document has a very small diameter of 100 to 500 μm, which is problematic in that it is not practical from the standpoint of handling and transplantation. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. WO2005 / 079728 [Patent Document 2] International Publication No. WO2008 / 120720 [Patent Document 3] Patent No. 4884678 [Patent Document 4] JP 2005-270647 A Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in consideration of such problems, and aims to provide a dental composition and a method for culturing dentin that are suitable for dental pulp regeneration treatment and that easily ensure the safety of the substances used. [Means for solving the problem]

[0009] The dental composition according to the present invention has the following composition. [1] Crushed human teeth The material has been demineralized. A dental composition used to promote dentin regeneration, comprising: [2] The dental composition according to [1], wherein the pulverized material has a particle size of 500 to 2000 μm. [3] A method for producing a dental composition used to promote dentin regeneration, comprising pulverizing human teeth to produce a pulverized material and then demineralizing the pulverized material. [4] The demineralization treatment is carried out using a strong acid. Used as a decalcification solution [3] The dental composition according to claim 1 Manufacturing method. [5] The demineralization treatment is carried out using 1 to 30% by weight of An aqueous solution of ethylenediamine derivative was used as the decalcification solution. [3] The dental composition according to claim 1 Manufacturing method. [6] The pulverized product Before the demineralization treatment Then, the sample is immersed in a solution containing an antibacterial agent and then dried. [3] The dental composition according to claim 1 Manufacturing method. [7] The dental composition according to [6], wherein the solution containing the antibacterial agent is an alcohol solution containing silver ions. Manufacturing method.

[0010] The dentin culture method according to the present invention has the following configuration. [8] Dental pulp cells and human tooth powder The material has been demineralized. A method for culturing dentin, comprising contacting dentin with a dental composition comprising the above-mentioned compound and inducing odontoblasts. Effect of the Invention

[0011] The present invention uses ground human teeth as an ingredient for promoting dentin regeneration, and therefore can provide a dental composition that can easily ensure safety and is suitable for use in dentin regeneration therapy to repair and regenerate covering dentin on the regenerated pulp tissue surface during pulp regeneration therapy or on the pulp surface during direct pulp capping and vital pulp resection therapy. [Brief description of the drawings]

[0012] [Figure 1] Schematic diagram showing the location of dental fragments in the treatment of a pulp-removed tooth [Figure 2A] CBCT image of Example 1 [Figure 2B] CBCT image showing an enlargement of area A in Figure 2A [Figure 3A] Photograph of tissue specimen in Example 1 [Figure 3B] A magnified photograph of area B in Figure 3A. [Figure 4A]Schematic diagram of dental pulp regeneration treatment using a dental composition containing ground tooth material immediately after transplantation. [Figure 4B] Schematic diagram of dental pulp regeneration treatment using a dental composition containing ground tooth material at an advanced stage of dentin regeneration. [Diagram 5] Schematic diagram explaining the process after dental pulp transplantation in dental pulp regeneration therapy [Figure 6] FIG. 1 is a schematic diagram showing the results at 4 to 6 months and 12 months after transplantation, and an explanatory diagram showing the correspondence between the tissue specimen photographs in Example 1. [Figure 7A] Micrograph of Example 2 (demineralized, 3% by weight EDTA) [Figure 7B] Micrograph of Example 2 (demineralized, 3% by weight EDTA) [Figure 7C] Magnified micrograph of region C in Figure 7B [Figure 8] Micrograph of Example 2 (Saline) [Figure 9A] Micrograph of Example 3 (decalcification treatment, 0.6N hydrochloric acid) [Figure 9B] Micrograph of Example 3 (demineralized, 17% by weight EDTA) [Figure 9C] Micrograph of Example 3 (decalcification treatment, 0.6N hydrochloric acid, 17% by weight EDTA) [Figure 10] Micrograph of Example 3 (undecalcified) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] <Dental Composition> The dental composition of the present invention (hereinafter, also referred to as "composition" as appropriate) contains pulverized human teeth (natural teeth). The cross section of a tooth contains dentin and dentinal tubules, which are suitable as a scaffold for dental pulp cells including dental pulp stem cells. Therefore, by using pulverized teeth, dentin regeneration can be promoted. The dental pulp stem cells and pulverized teeth used for dentin regeneration are preferably derived from the patient himself / herself in terms of compatibility, but may be provided by another person. By using dental pulp stem cells and pulverized teeth provided by a donor other than the patient, it is possible to promote the regeneration of dentin in the teeth of, for example, a patient who does not have a useless tooth such as a wisdom tooth or a patient whose function of dental pulp stem cells has decreased due to aging. Examples of persons other than the patient include the patient's relatives, but may be persons other than relatives as long as they are compatible with the patient. The dental pulp stem cells and pulverized teeth provided by a person other than the patient are preferably derived from the same tooth, but may be derived from different teeth. In the following, an embodiment of regenerating dentin of a tooth that has been extracted using dental pulp stem cells extracted from a useless tooth of a patient and pulverized teeth of the useless tooth will be described.

[0014] Dental pulp regeneration therapy begins with the extraction of the patient's unused tooth. Dental pulp stem cells are cultured after the extraction of pulp tissue from within the tooth, but traditionally the tooth chips remaining after the extraction of pulp tissue were discarded. Some of the dental pulp cells, including dental pulp stem cells, survive by adhering to the scaffold, and differentiate into odontoblasts in response to microstructures on the order of μm. In addition, the dentin and dentinal tubules of the tooth chips from which human dental pulp cells have been extracted are composed of the patient's own proteins. For this reason, it is believed that the tooth chips remaining after the extraction of pulp tissue are compatible with dental pulp stem cells and will not cause an immune rejection reaction.

[0015] Therefore, the inventors came up with the idea of ​​using the above-mentioned properties of the ground tooth material to use teeth that have been discarded in the past for inducing differentiation of dental pulp stem cells into odontoblasts and promoting dentin regeneration and repair. They also found that in order to achieve the above-mentioned induction and promotion functions, it is effective to keep the indexed teeth at an appropriate size and in an appropriate position. Furthermore, since teeth remaining after harvesting human dental pulp cells have been discarded in the past, dental compositions can be produced at low cost by using teeth from which human dental pulp cells have been harvested as a component for promoting dentin regeneration.

[0016] A tooth from which human dental pulp cells have been harvested has good compatibility with dental pulp stem cells extracted as dental pulp tissue from the tooth. Furthermore, proteins in dentin and dentinal tubules are unique to each individual patient. Therefore, by using a tooth from which human dental pulp cells have been harvested for dentin regeneration, it is possible to avoid immune rejection reactions and efficiently induce differentiation of human dental pulp cells into odontoblasts and regenerate dentin.

[0017] In the present invention, teeth that have been discarded in the past (unused teeth) are used to induce and promote dentin regeneration, since they are no longer needed after human dental pulp cells are harvested. Examples of teeth that are no longer needed include teeth extracted during orthodontic treatment and wisdom teeth. Since the composition of the present invention uses teeth that have been discarded in the past, it has little impact on the environment and can be produced by a very inexpensive and eco-friendly production method. In addition, tooth fragments, i.e., pulverized material, can be produced simultaneously with the dental pulp tissue using a pressing machine that splits the tooth and extracts the dental pulp tissue, so production efficiency is good.

[0018] Fig. 1 is a schematic diagram showing the position of the pulverized material 3 in the composition 4 in the treatment of a tooth that has had its pulp removed. As shown in the figure, in the treatment of a tooth that has had its pulp removed, a mixture 1 of dental pulp stem cells and a drug is transplanted into the tooth after the pulp is removed, and the pulverized tooth material 3 containing the composition 4 is placed on the mixture 1. After being covered with the dentin substitute 5, the part where the dentin and enamel were removed during the treatment of the crown part is reinforced with a cap of resin 2.

[0019] Odontoblasts created outside of the body are terminally differentiated cells, so when transplanted, they do not adhere to the root canal wall. The dental pulp stem cells in Mixture 1 are not terminally differentiated cells, but function as dentin matrix. Therefore, when transplanted into a living body, the dental pulp stem cells can adhere to the root canal wall.

[0020] As the dentin substitute 5, for example, a bioceramic material such as Biodentine (registered trademark, manufactured by Septodon), MTA (Mineal Trioxide Aggregate) cement, or the like is used.

[0021] Composition 4 has a predetermined particle size for the pulverized material 3 placed on top of mixture 1 in order to prevent the pulverized material 3 from moving downwards in mixture 1, i.e., away from resin 2. This allows the pulverized material 3 to be held in the upper crown of the mixture 1. By maintaining the position of the pulverized material 3 in the tooth after pulpectomy near the boundary between mixture 1 and resin 2, the effect of promoting dentin regeneration can be enhanced.

[0022] From the viewpoint of inducing differentiation into odontoblasts and regenerating dentin while keeping the pulverized material 3 in an appropriate position, it is preferable that the particle size of the pulverized material 3 is larger than the diameter of the root canal. However, the diameter of the root canal varies depending on the part of the tooth and the age of the patient. As the patient ages, the proportion of dentin increases, and the diameter of the pulp cavity filled with pulp tissue and the root canal narrows, resulting in a smaller diameter of the root canal. Therefore, the particle size of the pulverized material 3 used for inducing regeneration of dentin needs to be determined depending on the part of the tooth and the age of the patient.

[0023] As described above, the size of the root canal diameter r varies depending on the part of the tooth and the age of the patient, but is usually about 0.5 to 1.0 mm (500 to 1000 μm). Therefore, from the viewpoint of preventing the pulverized material 3 from falling into the root canal, the particle size of the pulverized material 3 should be 500 μm or more, preferably 600 μm or more, and more preferably 700 μm or more.

[0024] The particle size of the pulverized material 3 should be smaller than the width (cross-sectional distance) R of the portion where the mixture 1 of dental pulp stem cells and drug is injected, for example, 2000 μm or less. In addition, by reducing the particle size, the surface area of ​​the pulverized material 3 increases, which is advantageous for regenerating dentin. For this reason, the particle size of the pulverized material 3 is preferably 1500 μm or less, and more preferably 1000 μm or less.

[0025] As described above, in normal cases, the particle size of the pulverized material 3 is set to 500 to 2000 μm, preferably 500 to 1000 μm, so that the pulverized material 3 can be maintained at a position effective for promoting dentin regeneration. However, since the size of the root canal diameter varies depending on the individual and the site, the appropriate particle size is determined depending on the target root canal to be transplanted. From the viewpoint of promoting dentin regeneration, the particle size of the pulverized material 3 is preferably equal to or larger than the root canal diameter r of the root canal to be transplanted. For example, about 5 to 15 grains of the pulverized material 3 may be used for one tooth. In the present invention, the numerical range A to B means A or more and B or less.

[0026] The ground material 3 having a particle size within the above range can be, for example, one that is removed from the ground teeth using a strainer (sieve). The average particle size (median diameter, D50) of the ground material 3 contained in the composition 4 is preferably 600 to 1500 μm, more preferably 700 to 900 μm.

[0027] The pulverized material 3 includes an enamel portion and a dentin portion, but it is mainly the dentin portion that has the effect of promoting dental pulp regeneration. From the viewpoint of promoting dentin regeneration, it is preferable that the pulverized material 3 is demineralized. The effect of regenerating dentin by the dentin portion is improved by dissolving calcium salt crystals from the enamel portion of the pulverized material 3 by demineralization. In the present invention, demineralization refers to dissolving calcium phosphate, which is the main component of tooth enamel.

[0028] The demineralizing solution used in the demineralizing treatment of the pulverized material 3 may be an acidic to weakly alkaline aqueous solution in which a substrate is dissolved. Acid demineralization is a relatively strong treatment that utilizes the action of dissolving calcium phosphate. Substrates used in acid demineralization include hydrochloric acid, formic acid, nitric acid, trichloroacetic acid, sodium sulfate, sodium citrate, Planck-Lucro solution (a mixture of hydrochloric acid, formic acid, and aluminum chloride), and Morse's solution (a mixture of formic acid and sodium citrate). From the viewpoint of sufficiently dissolving calcium phosphate, a demineralization solution containing a strong acid such as hydrochloric acid or nitric acid as a substrate is preferable.

[0029] Demineralization using a chelating agent is a relatively weak treatment that utilizes the softening effect of calcium phosphate. Examples of substrates used in demineralization using a chelating agent include ethylenediamine derivatives such as sodium salt of ethylenediaminetetraacetic acid (EDTA). The ethylenediamine derivative is used, for example, as an aqueous solution of about 1 to 30% by weight.

[0030] From the viewpoint of sufficiently dissolving the enamel of the pulverized tooth to expose the dentin and promoting the differentiation of dental pulp stem cells into tubular dentin, bone-like dentin, etc. in the tooth after pulp extraction, a demineralizing solution for demineralization using an acid is preferable, and a strong acid such as hydrochloric acid is more preferable. The concentration of the aqueous solution of the strong acid is preferably 0.1 to 2.0 normal, more preferably 0.3 to 1.0 normal.

[0031] The time for treating the pulverized material with the demineralizing solution should be long enough to dissolve the enamel of the pulverized material. For example, when pulverized material 3 having a particle size (D50) of about 700 to 900 μm is demineralized using an aqueous solution of 0.3 to 1.0 normal hydrochloric acid, the treatment time is preferably 10 to 40 hours, more preferably 15 to 30 hours.

[0032] The pulverized material 3 is preferably one that has been immersed in a solution containing an antibacterial agent and then dried. Examples of the solution containing an antibacterial agent include an alcohol solution containing silver ions, pure water containing silver ions, and silica water.

[0033] The composition of the present embodiment can be produced, for example, as follows. (1) The tooth is placed in a tube with an open top, and the body is inserted from the top to randomly crush the tooth. The material used to break the tooth is a hard, biocompatible metal that will not cause any problems if it adheres to the tooth fragments. Examples of such metals include stainless steel, SUS, and titanium alloys. (2) Use a strainer to remove crushed tooth material (fragments) of a specified diameter (500 to 2000 μm). (3) Crushed tooth pieces of a specified size are transferred to a tube or the like and sterilized by soaking them in disinfectant ethanol. (4) Add pure water, etc., and confirm sterility by a culture test. Then soak in disinfectant ethanol again. (5) Allow the disinfectant ethanol to dry in a sterile space.

[0034] <Dentin culture method> The dentin culture method of the present invention induces odontoblasts by contacting dental pulp cells with a dental composition containing ground human teeth. Since no drug is used to induce odontoblasts, safety is easily ensured without causing anxiety about drugs in patients to whom the cultured dentin is applied. In addition, the ground tooth used to induce odontoblasts can be produced at the same time as the dental pulp is extracted, so dentin culture can be performed at low cost. EXAMPLES

[0035] [Example 1] Crushed dog teeth were transplanted into the root canals of two dogs (total of eight root canals), leaving part of the dental pulp intact. To observe the progress after transplantation, dental CT (cone beam CT, CBCT) images were taken four months after transplantation, and tissue samples were prepared and photographed six months after transplantation.

[0036] <Experimental conditions> Crushed tooth material: The teeth extracted from the dogs to be transplanted were crushed in a press, and crushed material (undecalcified) with particle sizes of 500 to 1000 μm was extracted using a strainer. Teeth to be transplanted: Teeth with vital pulp (remaining pulp) were selected as the transplant candidates. Transplant site: Without using any scaffolding material other than the crushed tooth material, 5 to 10 crushed pieces were placed near the entrance of the root canal of the tooth to be transplanted.

[0037] <Result> Figure 2A is a CBCT image taken 4 months after implantation, and Figure 2B is an enlarged CBCT image of the rectangular area A in Figure 2A. As shown in these figures, white, opaque dentin-like hard tissue was formed in the teeth implanted with the crushed material 4 months after implantation. In addition, dentin-like hard tissue was formed in all teeth implanted with the crushed material 4 months after implantation, similar to the teeth shown in Figures 2A and 2B.

[0038] FIG. 3A is a photograph of a tissue specimen six months after implantation, and FIG. 3B is a photograph of a tissue specimen showing an enlarged view of the area B enclosed by a rectangle in FIG. 3A. As shown in FIG. 3A, six months after implantation, a layer of bone-like dentin-like hard tissue was formed over a wide area on the regenerated dental pulp surface. In addition, as shown in FIG. 3B, it was found that regeneration of bone-like dentin was progressing between the root canal walls. These results indicate that the tooth pulverized material 3 placed near the entrance of the root canal can promote dentin regeneration.

[0039] FIG. 4A is a cross-sectional view showing a tooth at the time when the pulverized material is implanted, and FIG. 4B is a cross-sectional view showing a tooth at the time when sufficient time for regeneration has passed after implantation. As shown in FIG. 4A, by using pulverized material with a particle size of 500 to 1000 μm, regenerated dentin 6 can be formed near the entrance of the root canal. As the formation of the regenerated dentin 6 progresses, the regenerated dentin 6 fills the gap between the pulverized material and the remaining dentin of the crown, and the natural tooth and the regenerated dentin fuse together, forming a natural core (retaining part) under the resin 2. Therefore, the strength of the tooth after treatment is improved, and it is possible to prevent bacteria from reinfecting (microleakage) from the oral cavity and causing caries again (secondary caries).

[0040] The pulverized material 3 that promotes the formation of regenerated dentin 6 is made by pulverizing teeth. The pulverized material 3 is denatured and melted, and gradually replaced by regenerated dentin, so that after the regenerated dentin 6 is formed, it shows the same mechanical and chemical properties as the regenerated dentin 6. For example, the thermal expansion change caused by the change in environmental temperature is also the same. Therefore, no gap is generated between the pulverized material 3 and the regenerated dentin 6 or natural dentin when hot or cold food is eaten. In contrast, when an artificial material is used, there is a risk of gaps being generated due to differences in thermal expansion when hot or cold food is eaten. Since autologous proteins do not cause immune rejection reactions, it is more preferable to form the pulverized material 3 using the teeth of the same person as the treatment subject.

[0041] Figure 5 is a schematic diagram of a root canal model to explain the process after transplantation. As shown in the figure, a mixture 1 of dental pulp stem cells and a drug is transplanted into a root canal where a pulp has been removed, and then a crushed material 3 is placed on top of it, and then it is covered with resin 2. In the figure, a dentin substitute 5 is placed between the crushed material 3 and the resin 2.

[0042] Four to six months after transplantation, regenerated dentin (regenerated bone-like dentin) 6 is formed in the area where the crushed material 3 was left, and regenerated dental pulp 7 is formed in the area where the mixture 1 was transplanted. By regenerating not only dental pulp stem cells but also dental pulp tissue such as blood vessels and nerves and attracting dental pulp tissue to the area where dentin is being regenerated, dentin regeneration can be achieved in which cells attach to the sides of the root canal.

[0043] Twelve months after implantation, dentin tubules 8 are formed below the regenerated dentin 6 (above the regenerated pulp 7) (see "Dentin induction by implants of autolyzed antigen-extracted allogenic dentin on amputated pulps of dogs," Misako Nakashima, 1989).

[0044] 6 is an explanatory diagram showing the correspondence between the root canal models 4 to 6 months and 12 months after transplantation and the tissue specimen photograph of Example 1 (6 months after transplantation). As shown in the figure, the tissue specimen photograph 6 months after transplantation shows that regenerated dental pulp 7 has been formed. The photograph also shows that tubular dentin 8 has begun to form below regenerated dentin 6.

[0045] [Comparative Example 1] The crushed tooth from a dog was transplanted into a root canal with a part of the dental pulp remaining, and the progress was observed in the same manner as in Example 1, except that the crushed tooth with a particle size of 150 to 500 μm was used instead of the crushed tooth with a particle size of 500 to 1000 μm. However, when the crushed tooth with a particle size of 150 to 500 μm was used, no covering dentin formation was observed. This is thought to be because the crushed tooth did not remain near the entrance of the root canal, but fell to the apical side of the tooth.

[0046] The fact that the surface area of ​​the ground material increases by reducing the particle size is advantageous for promoting dentin regeneration. However, since no covering dentin formation was observed in the crown of the tooth, the location where the ground material is retained is important for promoting dentin regeneration, and it can be said that the ground material needs to be retained above the entrance of the root canal in the crown of the tooth.

[0047] In Example 1, the pulverized material 3 having a particle size of 500 to 1000 μm was used, and the pulverized material 3 remained near the entrance of the root canal, which promoted the formation of the covering dentin. In addition, the pulverized material having a particle size of 500 to 1000 μm was easier to handle than the pulverized material having a particle size of 150 to 500 μm, and was also superior in workability during transplantation.

[0048] [Examples 2 and 3] Of the enamel and dentin parts that make up tooth pulverized material, it is believed that the dentin part is the main contributor to the promotion of dentin regeneration. Therefore, the effect of treating the surface of the pulverized material with a solution was evaluated using the following method. Specifically, dental pulp stem cells were contacted with tooth pulverized material that had been subjected to different demineralization treatments, and the effect of promoting differentiation ability, i.e., dentin formation ability, was evaluated.

[0049] [Example 2] <Crushed teeth> Dog teeth were crushed using a press, and crushed tooth particles with particle sizes of 500 to 1000 μm were taken out using a strainer.

[0050] <Solution and treatment time> The ground material was immersed in the following solutions for the following times, washed with water, and then dried. Treatment (A) is a demineralization treatment in which enamel is removed from the ground material surface. Treatment (B) is a control. (A) 3% by weight EDTA aqueous solution (Smearclean; product name, Nippon Dental Pharmaceuticals, decalcification solution), 30 minutes (B) Saline, 30 min

[0051] <Culture> Dental pulp stem cells (1.25 × 10 6 The cells and dried tooth shavings were each made into pellets (cells and crushed material layered at the bottom of the tube), mixed with the following medium in a centrifuge tube, centrifuged, and cultured in the pelleted state in a centrifuge tube with an open lid. Culture medium: 10% fetal bovine serum (FBS) / Dulbecco's Modified Eagle's Medium (DMEM, Dulbecco's Modified Eagle's Medium) 2 ml Frequency of medium replacement: 3-4 days (to avoid damage to the pellet, replace 2 / 3 of the liquid volume at a time) Culture environment: 37℃ CO 2 Inside the incubator Culture period: 22 days

[0052] <Measurement> After culturing for approximately three weeks (22 days), the pelleted dental pulp stem cells and the crushed material were fixed in formalin and sliced, and the degree of dental pulp stem cell attachment to the tooth chips was confirmed by microscopic photographs.

[0053] [Example 3] <Crushed teeth> The unused teeth were crushed using a press, and crushed teeth with particle sizes of 750 to 1000 μm were removed using a strainer.

[0054] <Solution and treatment time> The crushed material was immersed in the following solutions for the following times, washed with water, and then dried. Treatments (C) to (E) are demineralization treatments, in which enamel is removed from the crushed material surface. Treatment (F) is the control. (C) 0.6N hydrochloric acid solution, 24 hours (D) 17% by weight EDTA aqueous solution (Smearclean; product name, Nippon Dental Pharmaceuticals, decalcification solution), 5 minutes (E) After process (C), process (D) (F) Undecalcified

[0055] <Culture> Dental pulp stem cells (4.7 × 10 5 The cells and dried tooth fragments were each pelleted and cultured, then mixed with the following medium in a centrifuge tube and centrifuged, and the pellets were then cultured in centrifuge tubes with loose lids. Culture medium: 2 ml of Dulbecco's modified Eagle medium (DMEM) containing 20% ​​by weight of Porcine serum and 0.5 mg / ml of L-ascorbic acid (= vitamin C, differentiation promoter) Frequency of medium replacement: 3-4 days (to avoid damage to the pellet, replace 2 / 3 of the liquid volume at a time) Culture environment: 37℃ CO 2 Inside the incubator Culture period: 14 days

[0056] <Evaluation of demineralization treatment> The crushed materials were divided into equal numbers into 35 mm dishes, and the difference in the degree of X-ray penetration due to demineralization was visually confirmed using a dental X-ray machine KDEX-SII (Kinki Roentgen Kogyo Co., Ltd.). As a result, it was confirmed that the crushed material (F) was visible on the X-ray photograph, the crushed materials (C) and (E) were not visible on the X-ray photograph, and the crushed material (D) was slightly visible on the X-ray photograph. From these results, it was confirmed that the enamel of the crushed materials (C) and (E) was sufficiently dissolved by the demineralization treatment, and dentin became the main component, and that a small amount of enamel remained in the crushed material (D) even after the demineralization treatment.

[0057] <Measurement> After culturing for approximately two weeks (14 days), the pelleted dental pulp stem cells and the crushed material were fixed in formalin and sliced, and the degree to which the dental pulp stem cells had attached to the tooth chips was confirmed using microscopic photographs.

[0058] Figures 7A to 7C and 8 are micrographs of the pulverized material and dental pulp stem cells that were pelletized and cultured after their surfaces were treated with (A) a 3 wt % EDTA aqueous solution and (B) a saline solution as a control in Example 2. As shown in Figures 7A to 7C, the pulverized material treated with 3 wt% EDTA aqueous solution had better adhesion of dental pulp stem cells to the pellet surface than the pulverized material treated with the control physiological saline solution. The direction of the double-sided arrow in Figure 7A indicates the elongation direction of the dentinal tubules, i.e., the tubular structure of dentin (pore diameter 1 to 2 μm). Figure 7C is an enlarged view of area C in Figure 7B.

[0059] From the photographs in FIG. 7A to FIG. 7C, it was found that the adhesiveness between the pulverized material and dental pulp stem cells was improved by subjecting the pulverized material to a demineralization treatment.

[0060] Figures 9A, 9B and 9C are micrographs of the pellets of the pulverized material treated with (C), (D) and (E) and dental pulp stem cells cultured in Example 3. Figure 10 is a micrograph of the pellets of the untreated pulverized material and dental pulp stem cells cultured as a control.

[0061] Dental pulp stem cells are adhesive cells that grow in a star-like shape when they are single cells. However, when dental pulp stem cells become dense, their cell shape elongates and promotes the formation of tissue. This elongation of tissue cells is a characteristic of dental pulp stem cells. When cultured in pellet form, the culture environment is dense, and if the ground surface is an appropriate adhesive scaffold, dental pulp stem cells will elongate in shape, become directional, and differentiate into tissue.

[0062] In the micrographs of Figures 9A and 9C, it can be seen that the cells are elongated and directional, and that the cells are aligned with each other. In contrast, in the micrograph of Figure 9B, the directionality of cell elongation and the alignment of cells cannot be clearly seen. From these results, it can be said that in order to promote the differentiation of dental pulp stem cells into tubular dentin, bone-like dentin, etc. in teeth after pulp extraction, it is effective to fully dissolve the enamel of the pulverized material by demineralization treatment and expose dentin on the surface of the pulverized material.

[0063] In addition, when the pulverized material was treated with hydrochloric acid (0.6N) for 60 minutes, the dental pulp stem cells were peeled off from the pellet surface of the pulverized material, and the adhesion state was slightly inferior to that of the pulverized material treated with 3 wt% EDTA aqueous solution. From this, when using a 0.6N hydrochloric acid aqueous solution, it is preferable to perform the demineralization treatment for a time longer than 60 minutes, for example, 10 hours or more, 15 hours or more, or 20 hours or more, from the viewpoint of sufficiently dissolving the enamel on the surface of the pulverized tooth material.

[0064] As shown in Figure 8, when saline treatment with solution (B) was used as a control in Example 2, no effect of improving the adhesion state of dental pulp stem cells was observed, as was the case when the pulverized material was decalcified with solution (A). Furthermore, as shown in FIG. 10, in the undecalcified (F) pulverized material used as a control in Example 3, no directionality due to cell elongation or alignment of cells was observed. [Industrial Applicability]

[0065] The dental composition and dentin culture method of the present invention can be used in caries treatment, pulpectomy, and infected root canal treatment. [Explanation of symbols]

[0066] 1:Mixture 2: Resin 3: Crushed material 4: Composition 5: Dentin substitute 6: Regenerated dentin 7: Regenerative dental pulp 8: tubular dentin A~C: Area r: Root canal diameter R: Section distance

Claims

1. A dental composition used to promote the regeneration of dentin, characterized in that it contains ground human teeth that have been demineralized.

2. The particle size of the pulverized product is 500 to 2000 μm. The dental composition of claim 1 .

3. A method for producing a dental composition used to promote dentin regeneration, comprising pulverizing human teeth to form a pulverized material and demineralizing the pulverized material.

4. The demineralization treatment uses a strong acid as a demineralization solution. A method for producing the dental composition according to claim 3.

5. The demineralization treatment uses an aqueous solution of 1 to 30% by weight of an ethylenediamine derivative as a demineralization solution. A method for producing the dental composition according to claim 3.

6. Before the demineralization treatment, the pulverized material is immersed in a solution containing an antibacterial agent and then dried. A method for producing the dental composition according to claim 3.

7. The solution containing the antibacterial agent is an alcohol solution containing silver ions; A method for producing the dental composition according to claim 6.

8. A method for culturing dentin, comprising contacting dental pulp cells with a dental composition containing demineralized pulp of human teeth to induce odontoblasts.

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