JP2005341961A - Method of inducing human pulp cell differentiation and composition for dentin regeneration - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of inducing the differentiation of a human pulp cell to an odontoblastic cell by which a dental disease patient such as the one suffering from tooth decay can be treated; and to provide a method of regenerating the dentin. <P>SOLUTION: The method of producing the odontoblastic cell the differentiation into which has been induced involves culturing at least one of the isolated human pulp cell and the cell capable of differentiating into the human human pulp cell in the presence of 1,25-(dihydroxy)vitamin D<SB>3</SB>, dexamethasone and β-glycerophosphate. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a method for inducing differentiation from human dental pulp cells to odontoblasts, a method for regenerating dentin, and a composition for regenerating dentin. More specifically, the present invention differentiates human dental pulp cells into odontoblasts by culturing them in the presence of 1,25 (dihydroxy) vitamin D ₃ , dexamethasone and β-glycerophosphate, The present invention relates to a method for regenerating dentin by culturing and / or transplanting together. The present invention relates to a dentin regeneration composition obtained by the above culture. The present invention further relates to dentin regenerated by the above method. The present invention further relates to a method for treating a dental patient using the odontoblasts induced to differentiate by the above method or the regenerated dentin.

  The modern society is an aging society, and it is expected that about 20% of the Japanese population will be 65 years old or older in a few years. The majority of these elderly people have lost some or all of their teeth, and many have used dentures (so-called dentures). Patients have the general perception that conventional dentures have not only practical problems such as attachment and detachment, but also a feeling of wearing, but also a psychological symbol of aging. It is. Furthermore, it is known that when all teeth are lost, if a complete denture is attached, the masticatory ability is about one-fifth that of a normal natural tooth. Furthermore, it has become clear that masticatory stimulation to the brain has an effect of preventing dementia, and that a decrease in masticatory power promotes dementia.

  Caries and periodontal diseases are cited as reasons for tooth loss. Root canal treatment is widely used as a treatment for preserving teeth that have pulpitis or pulpal necrosis due to severe caries. This is a treatment that removes dental pulp tissue infected by caries and fills it with an artificial drug. However, the root canal cannot be completely sealed because it has a complex shape. If the bacteria in the root canal and dentinal tubules have not been completely removed, secondary infection must be performed and treatment must be performed again, and it is said that the life of a tooth subjected to root canal treatment is short. In order to maintain the tooth for a long period of time, it is necessary to preserve the pulp as much as possible and increase the success rate of root canal treatment. In order to solve these problems, it is possible to completely seal the natural dentin by regenerating natural dentin, closing the exposed medulla by medullary cavity perforation and caries treatment, and root canal treatment. It can also be used as a filler.

  A method for regenerating natural dentin and using it for treatment has been proposed and studied. For example, Non-Patent Document 1 describes a sample of cultured human dental pulp cells seeded on a powder of a complex of hydroxyapatite (hereinafter abbreviated as HAp) and tricalcium phosphate (hereinafter abbreviated as β-TCP). It has been disclosed to be removed 6 weeks after transplantation subcutaneously. Then, the formation of hard tissue was confirmed from the hematoxylin / eosin stained image after extraction, and when RNA extracted from this sample was evaluated using RT-PCR, mRNA of the odontoblast differentiation marker was expressed. That is, it is disclosed that the obtained hard tissue was a dentin-like tissue. However, the amount of hard tissue formed is very small, and a larger amount of dentine regeneration is required for clinical application.

Furthermore, Non-Patent Document 2 shows that the addition of 1,25- (dihydroxy) vitamin D ₃ and TGF-β to human dental pulp cells increases alkaline phosphatase activity, which is an indicator of hard tissue formation. Yes. However, the activity of alkaline phosphatase in Non-Patent Document 2 is insufficient, and peptide factors such as TGF-β are expensive and have a problem in supply, and are rapidly deactivated or have an optimum concentration. Is not a satisfactory method in practical use.

S. Gronthos et al., Proc. Natl. Acad. Sci. USA, 2000, 97 (25): 13625-13630 P. Pavasant et al., Archives of Oral Biology, 2003, 48, 717-722

  An object of the present invention is to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a method for more efficiently differentiating human dental pulp cells into odontoblasts that form dentin. Moreover, this invention made it the problem which should be solved to provide the method of reproducing | regenerating as much dentin as possible for clinical application to dental diseases, such as a caries. Furthermore, another object of the present invention is to provide a method for treating dental diseases such as caries using differentiated odontoblasts or regenerated dentin.

As a result of intensive studies to solve the above problems, the present inventors have determined that human dental pulp cells or cells that can differentiate into human dental pulp cells are present in the presence of 1,25 (dihydroxy) vitamin D ₃ , dexamethasone and β-glycerophosphate. It was proved that the cells can be efficiently differentiated into odontoblasts that form dentin by culturing the cells. The present invention has been completed based on these findings.

That is, according to the present invention, at least one of the isolated human dental pulp cells or cells that can differentiate into human dental pulp cells is cultured in the presence of 1,25 (dihydroxy) vitamin D ₃ , dexamethasone and β-glycerophosphate. And a method for producing differentiation-induced odontoblasts.

Preferably, the cells are cultured on a carrier.
Preferably, the concentration of 1,25 (dihydroxy) vitamin D _{3 in} the medium is 1 × 10 ⁻⁸ to 1 × 10 ⁻⁵ mol / l and the concentration of dexamethasone in the medium is 1 × 10 ⁻⁹ to 1 ×. 10 ⁻⁶ mol / l, and the concentration of β-glycerophosphate in the medium is 1 to 30 mmol / l.

Preferably, the method for producing differentiation-induced odontoblasts according to the present invention comprises (i) a step of culturing at least one of isolated human dental pulp cells or cells that can differentiate into human dental pulp cells, (ii) the cells And (iii) culturing the cells in a medium containing 1,25 (dihydroxy) vitamin D ₃ , dexamethasone and β-glycerophosphate.
Preferably, the carrier is a ceramic carrier.

According to another aspect of the present invention, a differentiation-induced odontoblast obtained by the above method is provided.
According to still another aspect of the present invention, there is provided a cell composition for dentin regeneration comprising the above-described differentiation-induced odontoblast.
According to still another aspect of the present invention, there is provided a method for regenerating dentin, comprising culturing differentiation-induced odontoblasts obtained by the above method.
According to still another aspect of the present invention, dentin regenerated by the above method is provided.

  According to still another aspect of the present invention, there is provided a method for treating a dental patient, comprising transplanting a differentiation-induced odontoblast obtained by the above method or a dentin regenerated by the above method to a patient. The

  By using the method of the present invention, after forming dentin on the medium, or transplanting odontoblasts induced to differentiate so as to form dentin, together with a carrier or the like as necessary, The dentin can be regenerated to fill the defect or root canal. As a result, it is possible to maintain the tooth, which is a very effective treatment with a low probability of secondary infection. In addition, since it can maintain its own teeth for a long time, it greatly contributes to improving the quality of life (QOL) of patients.

Hereinafter, embodiments of the present invention will be described in detail.
According to the method of producing differentiation induced odontoblast according to the present invention, at least one of human dental pulp cells or cells that can differentiate into human dental pulp cells is composed of 1,25 (dihydroxy) vitamin D ₃ , dexamethasone and β-glycerophosphate. It is characterized by culturing in the presence.

  In the present invention, human dental pulp cells or cells that can differentiate into human dental pulp cells may be cultured as a single cell composed of one type of cell, or may be cultured as a cell mixture composed of two or more types of cells. .

  Human dental pulp cells or cells that can differentiate into human dental pulp cells can be collected from human extracted teeth or the like. Human dental pulp cells can be collected according to the method described in, for example, About I., Other Experimental cell research, 258, 33-41, 2000. Moreover, the cell which can be differentiated into a human dental pulp cell can be extract | collected, for example with the following method. Aseptically remove the impacted teeth and store them in a suitable storage solution such as a Phosphate Buffered Saline (hereinafter abbreviated as PBS) solution. Remove the calcified portion of the teeth, cut the tissue into small pieces with a scalpel, and wash the tissue with a PBS solution or the like. Subsequently, it is preferable to enzyme-treat the tissue using collagenase or dispase. After the enzyme treatment, the cells can be collected by pipetting and centrifugation.

Cell culture can be performed using normal serum-containing medium or serum-free medium used for animal cell culture under normal animal cell culture conditions (eg, room temperature to 37 ° C; 5% CO ₂ incubator, etc.) Can be done below. Moreover, it is also possible to add additives, such as ascorbic acid, to a culture medium. Although the form of culture is not particularly limited, for example, it can be performed by static culture.

The addition amount of 1,25 (dihydroxy) vitamin D ₃ is 1 × 10 ⁻⁸ to 1 × 10 ⁻⁵ mol / l, more preferably 1 × 10 ⁻⁷ to 1 × 10 ⁻⁶ mol / l in the medium. is there.

Moreover, the addition amount of dexamethasone is 1 × 10 ⁻⁹ to 1 × 10 ⁻⁶ mol / l, more preferably 1 × 10 ⁻⁸ to 1 × 10 ⁻⁷ mol / l in the medium.

  Furthermore, the addition amount of (beta) -glycerophosphate is 1-30 mmol / l in a culture medium, More preferably, it is 2-10 mmol / l.

  In the present invention, cells may be cultured on a carrier or may be cultured without a carrier. As the carrier, a carrier that can endure the time required for the formation of dentin and is rapidly absorbed thereafter is preferable. Furthermore, it is preferable to use a carrier made of a material having high affinity for cells.

  The material of the carrier is not particularly limited as long as it satisfies the above characteristics. For example, ceramic materials (for example, hydroxyapatite (HAp), tricalcium phosphate (β-TCP), tetracalcium phosphate), poly Synthetic polymer materials such as glycolic acid (PGA), poly (DL-lactide-co-glycoside) (PLGA), polylactic acid (PLLA), polycaprolactone, or protein materials such as collagen, gelatin, fibrin, or hyaluronic acid and Naturally derived materials such as salts thereof, alginic acid and salts thereof, ivory and coral can also be used.

  In the case of using a synthetic material such as PGA, PLLA, PLGA, or polycaprolactone, the surface may be coated with a collagen solution or a fibronectin solution in order to enhance cell adhesion and proliferation.

  As the form of the carrier, a mesh form, a sponge form, a gel form, a non-woven form, a granular form, and the like are possible.

  The carrier is preferably processed into a shape in which cells can be easily transplanted, and is preferably a plate-like or spherical porous body or hollow, with one end being opened and a blood vessel easily entering from the periphery.

  The carrier is preferably prepared in a form suitable for the purpose. For this purpose, a mold is obtained using an impression material after a desired form is made of a resin. Thereafter, the desired form can be reproduced by taking out the resin mold and pouring the synthetic material constituting the carrier.

According to one embodiment of the present invention, the method of the present invention can include the following three steps.
(I) a step of culturing at least one of the separated human dental pulp cells or cells that can differentiate into human dental pulp cells (primary culture);
(Ii) a step of seeding and culturing the cells on a carrier (secondary culture); and (iii) culturing the cells in a medium containing 1,25 (dihydroxy) vitamin D ₃ , dexamethasone and β-glycerophosphate. Process (differentiation induction):

The above step (i) is a culture performed in order to obtain a number of cells necessary for regenerating dentin in the subsequent steps. The cell culture in the step (i) is performed using a normal serum-containing medium or a serum-free medium used for animal cell culture under normal animal cell culture conditions (for example, a temperature from room temperature to 37 ° C .; 5% CO ₂ Under the incubator). Moreover, it is also possible to add additives, such as ascorbic acid, to a culture medium. Although the form of culture is not particularly limited, for example, it can be performed by static culture. In addition, the cells obtained in step (i) can be stored frozen, and those that have been thawed and expanded as necessary can be used in the operations after step (ii). According to this method, it is not necessary to collect cells each time dentin regeneration is performed, and the burden on the patient can be reduced.

The above step (ii) is a step of growing cells on a carrier. The cells seeded on the carrier grow by adhering to the carrier. The cell culture in the step (ii) is also performed by using a normal serum-containing medium or serum-free medium used for animal cell culture under normal animal cell culture conditions (for example, a temperature from room temperature to 37 ° C .; 5% CO ₂ Under the incubator). The culture period is preferably 1 to 30 days, more preferably 5 to 25 days.

The above step (iii) is a step of promoting differentiation induction into odontoblasts. The amounts used of 1,25 (dihydroxy) vitamin D ₃ , dexamethasone and β-glycerophosphate are as described above in the present specification.

  In the method of the present invention, human dental pulp cells or cells that can differentiate into human dental pulp cells are seeded on a carrier, or if necessary, cultured after seeding, and the cultured cells can be directly transplanted to a patient together with the carrier. good. Alternatively, more preferably, isolated human dental pulp cells or cells that can differentiate into human dental pulp cells are cultured and expanded and then seeded on a carrier, or if necessary, cultured after seeding, and then the cultured cells are used as a carrier. Can be transplanted together to regenerate dentin in the body.

  Alternatively, isolated human dental pulp cells or cells that can be differentiated into human dental pulp cells are cultured and expanded and then seeded on a carrier or cultured after seeding if necessary, and then the cultured cells are combined with the carrier. It can be transplanted into a transplanted animal, and the dentin can be regenerated in the body of the transplanted animal. Although the kind of transplant animal is not specifically limited, Preferably it is a mammal, For example, rodent animals, such as a mouse | mouth (nude mouse etc.) and a rat (nude rat etc.), can be used. The site of transplantation is not particularly limited, and examples include subcutaneous dorsal part.

The odontoblasts induced to differentiate by the above-described method of the present invention and the dentin regenerated by the method of the present invention can be treated by transplanting them into a patient having a dental disease such as caries. . That is, application to a method for treating a dental disease using dentin, for example, perforation of the medullary cavity, treatment of the exposed pulp, root canal treatment, and the like is also within the scope of the present invention. The dentin can be formed by continuing the growth of the dentin even after being transplanted to the dental patient.
The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Comparative Example 1: Measurement of cell number and ALPase activity of cultured human dental pulp cells (conditions without added factor)
(1) Collection and preservation The wisdom of a patient (29-year-old female) who obtained informed consent by a doctor was used. The extracted wisdom tooth was scraped with a turbine to expose the pulp, and the pulp tissue was aseptically collected and stored in a PBS solution containing 10% antibiotic.

  The pulp tissue was cut into small pieces of about 2 mm with a scalpel and washed 5 times with PBS solution.

(2) Cell recovery
The washed tissue was enzymatically treated for 50 minutes using an enzyme solution in which 2 mg / ml collagenase was dissolved in DMEM medium. The resulting tissue was pipetted for 10 minutes using a 25 ml pipette. The 25 ml supernatant was centrifuged to recover the cells. The obtained cells were washed five times with a medium in which DMEM medium and Nutrient Mixture F-12 were mixed 1: 1 (hereinafter abbreviated as DMEM / F12) containing 15% serum, and then filtered through a 70 μm cell strainer. The cells were collected by centrifugation.

(3) Static culture Collected cells were cultured in DMEM / F12 medium containing 15% serum at a concentration of 100 μmol / l and cultured under conditions of 37 ° C. and 5% CO ₂ and subcultured twice. The cells were detached from the cell culture flask with trypsin-EDTA, seeded at 1 × 10 ⁴ cells / well in a 12-well plate, and then statically cultured under conditions of 37 ° C. and 5% CO ₂ .

(4) Measurement
For measurement of ALPase activity, p-Nitrophenyl phosphate tablets sets (manufactured by SIGMA) were used. After cell seeding, the culture supernatant of the plate after 7 days and 10 days has been removed by suction, washed with PBS solution, and then added 800 μl of paranitrophenyl phosphate (hereinafter abbreviated as PNPP) solution to each well. The reaction was carried out for 6 minutes at 37 ° C. and 5% CO ₂ . An equal amount of 0.2N NaOH solution was added to stop the reaction, 200 μl was added to a 96-well plate, and the absorbance at 415 nm was measured using a microplate reader (GENios manufactured by TECAN).

Cell Counting Kit-8 (manufactured by Dojindo Laboratories) was used for the measurement of the number of cells. After cell seeding, the culture supernatant of the plate after 7 days and 10 days is removed by aspiration, 1 ml of DMEM / F12 medium is added to each well, and 100 μl of Cell Counting Kit-8 solution is added to each well. The color reaction was carried out for 60 minutes under the conditions of 5 ° C. and 5% CO ₂ . The reagent solution after the reaction was diluted 4-fold with distilled water, 200 μl was added to a 96-well plate, and absorbance at 450 nm was measured using a microplate reader (GENios manufactured by TECAN).

(5) Results The measured results are shown in Fig. 1 (ind (-)).

Comparative Example 2: Measurement of cell number and ALPase activity of human dental pulp cells cultured with addition of TGF-β1 and 1,25 (dihydroxy) vitamin D ₃ (conditions described in Non-Patent Document 2)
In Comparative Example 2 below, (1) collection and storage, (2) cell recovery, and (4) measurement were performed under the same conditions as in Comparative Example 1, and only for static culture and results under different conditions. Detailed description.

Stationary culture Collected cells were cultured in DMEM / F12 medium containing 15% serum at a concentration of 100 μmol / l and cultured at 37 ° C under 5% CO ₂ , and cells passaged twice were trypsinized. After peeling from the cell culture flask with -EDTA, 1 × 10 ⁴ cells / well were seeded in a 12-well plate. Cultivation is carried out by using a DMEM / F12 medium containing 15% serum supplemented with 1 ng / ml TGF-β1, 10 ^-8 mol / l 1,25 (dihydroxy) vitamin D ₃ at 37 ° C and 5% CO _2. Was done.

Results The measurement results are shown in FIG. 1 (TGF + VD).

Comparative Example 3: Measurement of the number of cultured human dental pulp cells and ALPase activity (conditions without added factor)
(6) Collection and preservation The wisdom of a patient (24-year-old woman) who obtained informed consent by a doctor was used. The extracted wisdom tooth was scraped with a turbine to expose the pulp, and the pulp tissue was aseptically collected and stored in a PBS solution containing 10% antibiotic.

  The pulp tissue was cut into small pieces of about 2 mm with a scalpel and washed 5 times with PBS solution.

(7) Cell recovery
The washed tissue was enzymatically treated for 50 minutes using an enzyme solution in which 2 mg / ml collagenase was dissolved in DMEM medium. The resulting tissue was pipetted for 10 minutes using a 25 ml pipette. The 25 ml supernatant was centrifuged to recover the cells. The obtained cells were washed five times with a medium in which DMEM medium and Nutrient Mixture F-12 were mixed 1: 1 (hereinafter abbreviated as DMEM / F12) containing 15% serum, and then filtered through a 70 μm cell strainer. The cells were collected by centrifugation.

(8) Static culture Collected cells were cultured in DMEM / F12 medium containing 15% serum at a concentration of 100 μmol / l and cultured under conditions of 37 ° C. and 5% CO _{2 for} 4 passages. The cells were detached from the cell culture flask with trypsin-EDTA, seeded at 1 × 10 ⁴ cells / well in a 12-well plate, and then statically cultured under conditions of 37 ° C. and 5% CO ₂ .

(9) Measurement
For measurement of ALPase activity, p-Nitrophenyl phosphate tablets sets (manufactured by SIGMA) were used. After cell seeding, the culture supernatant of the plate after 7 days and 10 days has been removed by suction, washed with PBS solution, and then added 800 μl of paranitrophenyl phosphate (hereinafter abbreviated as PNPP) solution to each well. The reaction was carried out for 6 minutes at 37 ° C. and 5% CO ₂ . An equal amount of 0.2N NaOH solution was added to stop the reaction, 200 μl was added to a 96-well plate, and the absorbance at 415 nm was measured using a microplate reader (GENios manufactured by TECAN).

Cell Counting Kit-8 (manufactured by Dojindo Laboratories) was used for the measurement of the number of cells. After cell seeding, the culture supernatant of the plate after 7 days and 10 days is removed by aspiration, 1 ml of DMEM / F12 medium is added to each well, and 100 μl of Cell Counting Kit-8 solution is added to each well. The color reaction was carried out for 60 minutes under the conditions of 5 ° C. and 5% CO ₂ . The reagent solution after the reaction was diluted 4-fold with distilled water, 200 μl was added to a 96-well plate, and absorbance at 450 nm was measured using a microplate reader (GENios manufactured by TECAN).

(10) Results The measurement results are shown in FIG. 2 (ind (−)).

Comparative Example 4: Measurement of the number and ALPase activity of human dental pulp cells cultured with dexamethasone and potassium dihydrogen phosphate added (conditions described in Non-Patent Document 1) In the following Comparative Example 4, (6 The collection and storage, (7) cell recovery, and (9) measurement are performed under the same conditions as in Comparative Example 3, and only the static culture and the results under different conditions will be described in detail.

Stationary culture Collected cells were cultured in DMEM / F12 medium containing 15% serum at a concentration of 100 μmol / l and ascorbic acid at 37 ° C., 5% CO _2. After peeling from the cell culture flask with -EDTA, 1 × 10 ⁴ cells / well were seeded in a 12-well plate. For culture, 100 μmol / l ascorbic acid, 10 ⁻⁸ mol / l Dexamethasone, 2.9 mmol / l potassium dihydrogen phosphate, 20 mmol / l HEPES ([2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfone Using DMEM / F12 medium containing 15% serum supplemented with (acid), static culture was performed under conditions of 37 ° C. and 5% CO ₂ .

Results The measurement results are shown in FIG. 2 (Dex + KH ₂ PO ₄ ).

Comparative Example 5: Measurement of cell number and ALPase activity of human dental pulp cells cultured with β-glycerophosphate and dexamethasone added (conditions in which only 1,25 (dihydroxy) vitamin D ₃ is not added) In Comparative Example 5 below, , (6) Collection and storage, (7) Cell recovery, and (9) Measurement are performed under the same conditions as in Comparative example 3, and only the static culture and the results under different conditions will be described in detail.

Stationary culture Collected cells were cultured in DMEM / F12 medium containing 15% serum at a concentration of 100 μmol / l and cultured at 37 ° C under 5% CO ₂ , and cells passaged twice were trypsinized. After peeling from the cell culture flask with -EDTA, 1 × 10 ⁴ cells / well were seeded in a 12-well plate. For the culture, static culture was performed at 37 ° C. and 5% CO ₂ using a DMEM / F12 medium containing 15% serum supplemented with 10 mmol / l β-Glycerophosphate and 10 ⁻⁸ mol / l Dexamethasone.

Results The measurement results are shown in FIG. 2 (βGly + Dex).

Comparative Example 6: Measurement of the number of cultured human dental pulp cells and ALPase activity (conditions without addition factor)
(11) Collection and preservation The wisdom of a patient (22-year-old female) who obtained informed consent by a doctor was used. The extracted wisdom tooth was scraped with a turbine to expose the pulp, and the pulp tissue was aseptically collected and stored in a PBS solution containing 10% antibiotic.

  The pulp tissue was cut into small pieces of about 2 mm with a scalpel and washed 5 times with PBS solution.

(12) Cell recovery
The washed tissue was enzymatically treated for 50 minutes using an enzyme solution in which 2 mg / ml collagenase was dissolved in DMEM medium. The resulting tissue was pipetted for 10 minutes using a 25 ml pipette. The 25 ml supernatant was centrifuged to recover the cells. The obtained cells were washed five times with a medium in which DMEM medium and Nutrient Mixture F-12 were mixed 1: 1 (hereinafter abbreviated as DMEM / F12) containing 15% serum, and then filtered through a 70 μm cell strainer. The cells were collected by centrifugation.

(13) Static culture The collected cells were cultured in DMEM / F12 medium containing 15% serum at a concentration of 100 μmol / l at 37 ° C. under 5% CO ₂ condition, and subcultured twice. The cells were detached from the cell culture flask with trypsin-EDTA, seeded at 1 × 10 ⁴ cells / well in a 12-well plate, and then statically cultured under conditions of 37 ° C. and 5% CO ₂ .

(14) Measurement
For measurement of ALPase activity, p-Nitrophenyl phosphate tablets sets (manufactured by SIGMA) were used. After cell seeding, the culture supernatant of the plate after 7 days and 10 days has been removed by suction, washed with PBS solution, and then added 800 μl of paranitrophenyl phosphate (hereinafter abbreviated as PNPP) solution to each well. The reaction was carried out for 6 minutes at 37 ° C. and 5% CO ₂ . An equal amount of 0.2N NaOH solution was added to stop the reaction, 200 μl was added to a 96-well plate, and the absorbance at 415 nm was measured using a microplate reader (GENios manufactured by TECAN).

Cell Counting Kit-8 (manufactured by Dojindo Laboratories) was used for the measurement of the number of cells. After cell seeding, the culture supernatant of the plate after 7 days and 10 days is removed by aspiration, 1 ml of DMEM / F12 medium is added to each well, and 100 μl of Cell Counting Kit-8 solution is added to each well. The color reaction was carried out for 60 minutes under the conditions of 5 ° C. and 5% CO ₂ . The reagent solution after the reaction was diluted 4-fold with distilled water, 200 μl was added to a 96-well plate, and absorbance at 450 nm was measured using a microplate reader (GENios manufactured by TECAN).

(15) Results The measured results are shown in FIG. 3 (ind (-)).

Comparative Example 7: Measurement of cell number and ALPase activity of human dental pulp cells cultured with β-glycerophosphate and dexamethasone added (conditions in which only 1,25 (dihydroxy) vitamin D ₃ is not added) In the following Comparative Example 7, , (11) collection and storage, (12) cell recovery, and (14) measurement are performed under the same conditions as in Comparative Example 6, and only static culture and results under different conditions will be described in detail.

Stationary culture Collected cells were cultured in DMEM / F12 medium containing 15% serum at a concentration of 100 μmol / l and cultured at 37 ° C under 5% CO ₂ , and cells passaged twice were trypsinized. After peeling from the cell culture flask with -EDTA, 1 × 10 ⁴ cells / well were seeded in a 12-well plate. For the culture, static culture was performed at 37 ° C. and 5% CO ₂ using a DMEM / F12 medium containing 15% serum supplemented with 10 mmol / l β-Glycerophosphate and 10 ⁻⁸ mol / l Dexamethasone.

Results The measurement results are shown in FIG. 3 (βGly + Dex).

Example 1: Measurement of number and ALPase activity of human dental pulp cells cultured with addition of differentiation-inducing factor In Example 1 below, (1) collection and storage, (2) cell recovery, (4) measurement Is performed under the same conditions as in Comparative Example 1, and only the static culture and the results under different conditions will be described in detail.

Stationary culture Collected cells were cultured in DMEM / F12 medium containing 15% serum at a concentration of 100 μmol / l and cultured at 37 ° C under 5% CO ₂ , and cells passaged twice were trypsinized. After peeling from the cell culture flask with -EDTA, 1 × 10 ⁴ cells / well were seeded in a 12-well plate. DMEM / F12 medium containing 15% serum supplemented with 100 μmol / l ascorbic acid, 10 ⁻⁶ mol / l 1,25 (dihydroxy) vitamin D ₃ , 10 mmol / l β-Glycerophosphate, 10 ⁻⁸ mol / l Dexamethasone The static culture was performed under the conditions of 37 ° C. and 5% CO ₂ .

Results The measurement results are shown in FIG. 1 (VD + βGly + Dex).

  When the above Comparative Example 1 (conditions in which no additive factor is added), Comparative Example 2 (conditions described in Non-Patent Document 2) and Example 1 are compared, in the case of the conditions described in Example 1, cell differentiation proceeds. However, the ALPase activity, which is an index of the ability to differentiate into dental pulp cells and the ability to form dentin, is significantly increased (FIG. 1).

Example 2: Measurement of number and ALPase activity of human dental pulp cells cultured with addition of differentiation-inducing factor In Example 2 below, (11) collection and storage, (12) cell recovery, and (14) measurement Is performed under the same conditions as in Comparative Example 6, and only the static culture and the results under different conditions will be described in detail.

Stationary culture Collected cells were cultured in DMEM / F12 medium containing 15% serum at a concentration of 100 μmol / l and cultured at 37 ° C under 5% CO ₂ , and cells passaged twice were trypsinized. After peeling from the cell culture flask with -EDTA, 1 × 10 ⁴ cells / well were seeded in a 12-well plate. DMEM / F12 medium containing 15% serum supplemented with 100 μmol / l ascorbic acid, 10 ⁻⁶ mol / l 1,25 (dihydroxy) vitamin D ₃ , 10 mmol / l β-Glycerophosphate, 10 ⁻⁸ mol / l Dexamethasone The static culture was performed under the conditions of 37 ° C. and 5% CO ₂ .

Results The measurement results are shown in FIG. 3 (VD + βGly + Dex).

Comparing the above Comparative Example 3 (conditions in which no additive factor is added), Comparative Example 4 (conditions described in Non-Patent Document 1) and Comparative Example 5 (conditions in which only 1,25 (dihydroxy) vitamin D ₃ is not added) are compared. In Example 5, ALPase activity, which is an index of differentiation into dental pulp cells and dentin formation ability, is most increased (FIG. 2).

Similarly, when Comparative Example 6 (conditions in which no additive factor is added), Comparative Example 7 (conditions in which only 1,25 (dihydroxy) vitamin D ₃ is not added) and Example 2 are compared, in the case of the conditions of Example 2, the dental pulp ALPase activity, which is an index of cell differentiation and dentin formation ability, is significantly increased (FIG. 3). That is, the results of FIGS. 2 and 3 clearly show that the conditions of Example 2 are more effective than the conditions described in Non-Patent Document 1.

Comparative Example 8: Transplantation experiment not including the step of promoting differentiation induction into odontoblasts The wisdom of a patient (21-year-old male) who obtained informed consent by a doctor was used. The extracted wisdom tooth was scraped with a turbine to expose the pulp, and the pulp tissue was aseptically collected and stored in a PBS solution containing 10% antibiotic.

  The pulp tissue was cut into small pieces of about 2 mm with a scalpel and washed 5 times with PBS solution.

  The washed tissue was enzymatically treated for 50 minutes using an enzyme solution in which 2 mg / ml collagenase was dissolved in DMEM medium. The resulting tissue was pipetted for 10 minutes using a 25 ml pipette. The 25 ml supernatant was centrifuged to recover the cells. The obtained cells were washed 5 times with a medium containing 15% serum in DMEM / F12 medium, filtered with a 70 μm cell strainer, and centrifuged to collect the cells.

The recovered cells were cultured in DMEM / F12 medium containing 15% serum at a concentration of 100 μmol / l and cultured at 37 ° C. under 5% CO ₂ , and the first passaged cells were treated with trypsin-EDTA. After detaching from the cell culture flask, adjust the cell suspension to 5.0 × 10 ⁶ cells / 100 μl with DMEM / F12 medium containing 15% serum, and 130 mg of HAp / β-TCP porous material (Japan) using a 96-well plate. After sowing on a special ceramic industry, static culture was performed for 4.5 hours under conditions of 37 ° C. and 5% CO ₂ .

  After the cells adhered to the carrier, the cells were transferred to a 6-well plate and allowed to grow on the carrier for 13 days in a DMEM / F12 medium containing 15% serum.

  Nude mouse KSN was used as the transplanted animal. The dorsal skin of the nude mouse was incised, the carrier seeded with cells was placed subcutaneously, and the skin was sutured.

  Samples were collected 6 weeks after transplantation. The extracted sample was fixed with a 10% formalin solution and embedded in paraffin according to a conventional method to prepare a continuous tissue section. Thereafter, the sections were stained with hematoxylin-eosin (HE staining) and observed histologically.

  The graft removed 6 weeks after transplantation was maintained in its original form without being absorbed by the body. Moreover, as a result of observing the HE-stained tissue, a hard tissue was formed, but it was weak and the amount was small (FIG. 4).

Example 3: Transplantation experiment including a step of promoting differentiation induction into odontoblasts The wisdom of a patient (21-year-old male) who obtained informed consent by a doctor was used. The extracted wisdom tooth was scraped with a turbine to expose the pulp, and the pulp tissue was aseptically collected and stored in a PBS solution containing 10% antibiotic.

  The pulp tissue was cut into small pieces of about 2 mm with a scalpel and washed 5 times with PBS solution.

  The washed tissue was enzymatically treated for 50 minutes using an enzyme solution in which 2 mg / ml collagenase was dissolved in DMEM medium. The resulting tissue was pipetted for 10 minutes using a 25 ml pipette. The 25 ml supernatant was centrifuged to recover the cells. The obtained cells were washed 5 times with a medium containing 15% serum in DMEM / F12 medium, filtered with a 70 μm cell strainer, and centrifuged to collect the cells.

The recovered cells were cultured in DMEM / F12 medium containing 15% serum at a concentration of 100 μmol / l and cultured at 37 ° C under 5% CO ₂ , and the passaged cells were trypsin-EDTA. After detaching from the cell culture flask, adjust the cell suspension to 5.0 × 10 ⁶ cells / 100 μl with DMEM / F12 medium containing 15% serum, and 130 mg of HAp / β-TCP porous material (Japan) using a 96-well plate. After sowing on a special ceramic industry, static culture was performed for 4.5 hours under conditions of 37 ° C. and 5% CO ₂ .

  After the cells adhered to the carrier, the cells were transferred to a 6-well plate and allowed to grow on the carrier for 7 days in a DMEM / F12 medium containing 15% serum.

Furthermore, 100μmol / l ascorbic acid, 10 ⁻⁶ mol / l 1,25 (dihydroxy) vitamin D ₃ , 10 mmol / l β-Glycerophosphate, 10 ⁻⁸ mol / l Dexamethasone as a differentiation inducer in DMEM / F12 medium with 15% serum Cultured for 6 days in a medium added to the above, a composition for dentin regeneration was obtained.

  Nude mouse KSN was used as the transplanted animal. Nude mouse dorsal skin was incised, and the dentin regeneration composition obtained above was placed subcutaneously and the skin was sutured.

  Samples were collected 6 weeks after transplantation. The extracted sample was fixed with a 10% formalin solution and embedded in paraffin according to a conventional method to prepare a continuous tissue section. Thereafter, the sections were stained with HE and observed histologically.

  The graft removed 6 weeks after transplantation was maintained in its original form without being absorbed by the body. Moreover, as a result of observing the HE-stained tissue, a dentin-like hard tissue was remarkably formed as compared with Comparative Example 8 (FIG. 5).

FIG. 1 shows the number of cells, ALPase activity, and changes with time (7th day and 10th day) under the conditions of Comparative Example 1, Comparative Example 2 and Example 1. FIG. 2 shows the number of cells, ALPase activity, and changes with time (7th and 10th days) under the conditions of Comparative Example 3, Comparative Example 4 and Comparative Example 5. FIG. 3 shows the number of cells, ALPase activity, and changes with time (7th and 10th days) under the conditions of Comparative Example 6, Comparative Example 7 and Example 2. FIG. 4 shows an HE-stained image of a tissue obtained by transplanting cultured human dental pulp cells. FIG. 5 shows an HE-stained image (differentiation induction (+)) of a tissue obtained by transplantation of cultured human dental pulp cells.

Claims (10)

Differentiation-induced, comprising culturing at least one type of isolated human dental pulp cells or cells that can differentiate into human dental pulp cells in the presence of 1,25 (dihydroxy) vitamin D ₃ , dexamethasone and β-glycerophosphate A method for producing odontoblasts. The method of claim 1, wherein the cells are cultured on a carrier. The concentration of 1,25 (dihydroxy) vitamin D _{3 in} the medium is 1 × 10 ⁻⁸ to 1 × 10 ⁻⁵ mol / l, and the concentration of dexamethasone in the medium is 1 × 10 ⁻⁹ to 1 × 10 ^−6. The method according to claim 1, wherein the concentration is 1 mol / l and the concentration of β-glycerophosphate in the medium is 1 to 30 mmol / l. (I) a step of culturing at least one of the separated human dental pulp cells or cells that can differentiate into human dental pulp cells, (ii) a step of seeding and culturing the cells on a carrier, and (iii) 1 of the cells. , 25 (dihydroxy) vitamin D ₃ , culturing in a medium containing dexamethasone and β-glycerophosphate. The method of claim 4, wherein the support is a ceramic-based support. Differentiated ivory comprising culturing at least one of isolated human dental pulp cells or cells that can differentiate into human dental pulp cells in the presence of 1,25 (dihydroxy) vitamin D ₃ , dexamethasone and β-glycerophosphate Differentiated odontoblasts obtained by the method for producing blasts. A cell composition for dentin regeneration comprising the differentiation-induced odontoblast according to claim 6. A method for regenerating dentin, comprising culturing differentiation-induced odontoblasts obtained by the method according to claim 1. Differentiated ivory comprising culturing at least one of isolated human dental pulp cells or cells that can differentiate into human dental pulp cells in the presence of 1,25 (dihydroxy) vitamin D ₃ , dexamethasone and β-glycerophosphate A dentin regenerated by a method for regenerating dentin, comprising culturing differentiation-induced odontoblasts obtained by the method for producing blasts. A method for treating a dental patient, comprising transplanting differentiation-induced odontoblasts obtained by the method according to any one of claims 1 to 5 or dentin regenerated by the method according to claim 5 to a patient. .