JPWO2019216438A1 - Periodontal disease therapeutic agent containing culture supernatant of mesenchymal cells - Google Patents

Abstract

It is a periodontal disease therapeutic agent that can be used in combination with antibacterial treatment in the treatment of periodontal disease, has an excellent therapeutic effect in a short period of time, and regenerates periodontal tissue damaged by periodontal disease. The challenge is to provide a therapeutic agent for periodontal disease that can be promoted.
The present invention relates to a periodontal disease therapeutic agent containing a culture supernatant of mesenchymal cells, which is used in combination with antibacterial treatment against periodontal disease pathogens.
[Selection diagram] None

Description

The present invention relates to a periodontal disease therapeutic agent containing a culture supernatant of mesenchymal cells. In particular, the present invention relates to a periodontal disease therapeutic agent containing a culture supernatant of mesenchymal cells used in combination with an antibacterial treatment for the treatment of periodontal disease.

Periodontal disease affects more than 80% of the population, and is considered to be mainly caused by oral bacterial infection, increase in periodontal disease pathogens, bacterial invasion into tissues, and host immune response to infection. .. Periodontal disease An inflammatory disease in which the periodontal tissues (gingiva, cementum, periodontal ligament, and alveolar bone) that support the teeth are destroyed by infection with pathogenic bacteria. The disease involves the formation of periodontal pockets, tooth mobility, alveolar bone resorption, and drainage of the pockets, most of which progress painlessly. As periodontal disease progresses, not only inflammation and destruction of periodontal tissue but also bones that support teeth called alveolar bone are absorbed (or destroyed), and finally teeth fall out. As described above, periodontal disease is an extremely important disease that eventually leads to tooth loss and impairs the quality of life.

Since elimination of the source of infection is important for the treatment of periodontal disease, extremely primitive methods such as brushing and scaling and periodontal surgery such as sterilization by laser irradiation are still the mainstream. In addition, administration of tetracycline antibiotics has also been effective as a periodontal treatment.
It is known that specific periodontal groove gram-negative bacilli are involved in periodontitis. For example, adult periodontitis involves Bacteroides gingivalis, juvenile periodontitis involves Actinobacillus actinomycetemcomitans, and acute necrotizing ulcerative periodontitis involves Bacteroides intermedius. Tetracycline antibiotics have antibacterial activity against these periodontal disease pathogens. Minocycline is a kind of tetracycline antibiotic, shows a strong antibacterial action against the above-mentioned periodontopathogenic bacteria, and exerts an excellent clinical effect on periodontitis. For example, a method of directly administering an ointment containing minocycline to a periodontal pocket has been proposed, and as a periodontitis treatment drug, "Perioclin Dental Ointment" (sold by Sunstar Co., Ltd.) and "Periofeel Dental Ointment" (Showa) (Sold by Yakuhin Kako Co., Ltd.) is used clinically.

However, although administration of antibacterial agents and antibacterial treatment by periodontal surgery can suppress the progression of periodontal disease caused by periodontal pathogens, periodontal tissue that has been damaged once, such as patients with severe periodontal disease, In particular, the periodontal bone could not be regenerated.
In addition, while the use of antibiotics has a strong action and is excellent in effect, long-term use of antibiotics has problems such as the emergence of resistant bacteria and side effects, suggesting the limitation of drug therapy. Therefore, when antibiotics are used for the treatment of periodontal disease, it has been desired to realize a treatment policy that completes the treatment of periodontal disease in a short period of time while the amount of antibiotics used is limited.

Guided tissue regeneration (GTR) is known as one method for regenerating damaged periodontal tissue. The GTR method uses an absorbent or non-absorbable biofilm or titanium film to prevent periodontal tissue damage in order to prevent epithelial tissue that quickly covers the wound surface from infiltrating into the damaged part of the periodontal tissue. It is a method of arranging in the department. As a result, periodontal ligament-derived tissues and cells form new connective tissue adhesions on the wound surface with cementum formation. In the GTR method, since it is necessary to place the membrane in the living body, the operation time is prolonged and the surgical invasion is not a little, and postoperative complications and gingival recession become problems. Further, it has been reported that the cementum structure regenerated by the GTR method is different from the original cementum structure and that the running of collagen in the periodontal ligament is different (Non-Patent Document 1).
As another method, the Emdgain method is known. This method acts on the undifferentiated mesenchymal tissue in the periodontal ligament by applying Emdgain (registered trademark) containing enamel matrix derivative as the main component to the damaged part of the periodontal tissue after periodontal surgery. It differentiates into cementoblasts and creates acellular cementum. As a result, the destroyed cementum and the like are regenerated. For example, in Non-Patent Document 2, when a group of patients with vertical bone defects are treated with Emdgain, the periodontal pocket depth (PD) averages from 7.6 ± 1.3 mm to an average 8 months after the operation date. It is reported that it decreased to 3.5 ± 1.3 mm.

Further, in recent years, a method for regenerating damaged tissue using cells derived from a living body has been studied.
Patent Document 1 discloses the use of odontogenic stem cells in the preparation of products for treating periodontal disease.
However, cell therapy using cells guarantees problems related to infectious factors such as viruses, problems related to characterization and eligibility of cells used as raw materials, risk elimination of tumorigenicity, and efficacy, safety, and quality. However, there are many high hurdles in that a stable supply must be provided promptly to the medical field.
On the other hand, Patent Document 2 discloses a method for regenerating tissue damage using a culture supernatant of cells that does not contain the cells themselves. Specifically, we disclose a technique for culturing dental pulp stem cells and regenerating tissues using growth factors released from the cells. Patent Document 2 discloses an invention relating to a composition for treating an injured part, which comprises a stem cell culture supernatant obtained by serum-free culture of dental pulp stem cells.

Special Table 2017-507123 Japanese Unexamined Patent Publication No. 2016-65106

Shuichi Sato, "Current Status of Periodontal Disease Treatment Applying Regenerative Treatment", Nihon Univ. Dent J. 89, 93-99, 2015 Takeshi Sueda, Jiro Hoshino, Juno Yamamura, "Survey Report on Emdgain Usage Results in Multicenters", Periodontal Magazine, 46 (2): 152-160, 2004

It is a periodontal disease therapeutic agent that can be used in combination with antibacterial treatment in the treatment of periodontal disease, has an excellent therapeutic effect in a short period of time, and regenerates periodontal tissue damaged by periodontal disease. The challenge is to provide a therapeutic agent for periodontal disease that can be promoted.

As mentioned above, patients with severe periodontal disease have periodontal tissue damage, including alveolar bone resorption, and antibacterial treatments such as antibiotics in the treatment of periodontal disease may desire periodontal tissue regeneration. could not.
Therefore, the present inventors investigated a method capable of promoting the regeneration of periodontal tissue damaged by periodontal disease together with antibacterial treatment in the treatment of periodontal disease. Here, the method of directly administering the cell itself to the injured site as in Patent Document 1 has the problem of infectious factors such as viruses, the problem of evaluation / selection of the characteristics and eligibility of the cell itself used as a raw material, and the problem of the cell. There are still high hurdles to the problem of eliminating the risk of tumorigenicity and ensuring a certain level of quality in the cell preparation process. Therefore, the present inventors investigated whether only the culture supernatant of cells could be applied to the treatment of periodontal disease without using the cells themselves.
Patent Document 2, which discloses that the culture supernatant of cells is used for treatment, recovers growth factors from the culture supernatant by culturing human bone marrow stem cells (Lonza Co., ltd, USA) as an example, and the growth thereof. It describes that the periodontium was regenerated by injecting the factor into the damaged periodontal tissue.

Here, in the examples of Patent Document 2, a sample in which a two-walled periodontal defect is artificially created / imitated in the distal portion of the canine tooth of a dog and a sample having a periodontal defect inside the canine tooth of a woman are used. There is no disclosure as to whether the growth factor derived from human bone marrow stem cells is actually effective in the periodontal tissue site in the periodontal disease-affected state derived from the periodontal disease pathogen.
In addition, administration of cell culture supernatant in combination with conventional antibacterial treatment has not been investigated for patients suffering from periodontal disease.
On the other hand, the present inventors prepared a culture supernatant using dental pulp cells among the mesenchymal cells, and used the culture supernatant in combination with antibacterial treatment for patients suffering from severe periodontal disease. It was found that when administered, the score of periodontal disease by clinical examination was greatly improved in a short time, and the regeneration of periodontal tissue including alveolar bone, which could not be obtained by conventional antibacterial treatment, could be promoted. ..

The present invention has been completed based on the above findings and includes the following aspects:
In one aspect, the present invention
[1] The present invention relates to a periodontal disease therapeutic agent containing a culture supernatant of mesenchymal cells, which is used in combination with antibacterial treatment against periodontal disease pathogens.
Further, the periodontal disease therapeutic agent of the present invention, in one embodiment,
[2] The periodontal disease therapeutic agent according to the above [1], wherein the antibacterial treatment is selected from the group consisting of administration of an antibacterial agent, administration of a disinfectant solution, laser irradiation, scaling, root planing, and cleaning in a periodontal pocket. It is characterized in that it is at least one treatment to be performed.
Further, the periodontal disease therapeutic agent of the present invention, in one embodiment,
[3] The periodontal disease therapeutic agent according to the above [1] or [2].
The antibacterial treatment is characterized by administration of a periodontal disease therapeutic agent containing minocycline or a pharmaceutically acceptable salt thereof.
Further, the periodontal disease therapeutic agent of the present invention, in one embodiment,
[4] The periodontal disease therapeutic agent according to the above [3].
It is characterized by being used at the same time as administration of minocycline or a pharmaceutically acceptable salt thereof.
Further, the periodontal disease therapeutic agent of the present invention, in one embodiment,
[5] The periodontal disease therapeutic agent according to the above [3] or [4].
The amount of minocycline or a pharmaceutically acceptable salt thereof administered in combination with a periodontal disease therapeutic agent is less than 10 mg at a time.
Further, the periodontal disease therapeutic agent of the present invention, in one embodiment,
[6] The periodontal disease therapeutic agent according to any one of the above [1] to [5].
The culture supernatant of the mesenchymal cells is a culture supernatant obtained by culturing a group of mesenchymal cells including mesenchymal stem cells.
Further, the periodontal disease therapeutic agent of the present invention, in one embodiment,
[7] The periodontal disease therapeutic agent according to the above [6].
The mesenchymal cells are dental pulp cells, and the mesenchymal stem cells are dental pulp stem cells.
Further, the periodontal disease therapeutic agent of the present invention, in one embodiment,
[8] The periodontal disease therapeutic agent according to any one of the above [1] to [7].
It is characterized in that it is used in combination with at least one periodontal disease treatment selected from the group consisting of GTR method, β-TCP, bone graft material, collagen sponge, or b-FGF.
Further, the periodontal disease therapeutic agent of the present invention, in one embodiment,
[9] The periodontal disease therapeutic agent according to any one of the above [1] to [8].
It is characterized by being applied to periodontal tissue inflamed or damaged by periodontal disease.
Further, the periodontal disease therapeutic agent of the present invention, in one embodiment,
[10] The periodontal disease therapeutic agent according to any one of the above [1] to [9].
The culture supernatant of the mesenchymal cells is a culture supernatant obtained by culturing the mesenchymal cells using a serum-free medium.
Further, the periodontal disease therapeutic agent of the present invention, in one embodiment,
[11] The periodontal disease therapeutic agent according to the above [10].
The culture supernatant of the mesenchymal cells is a culture supernatant obtained by culturing the mesenchymal cells using a medium containing serum and subsequently culturing the mesenchymal cells using a serum-free medium. It is a feature.
Further, the periodontal disease therapeutic agent of the present invention, in one embodiment,
[12] The periodontal disease therapeutic agent according to any one of the above [1] to [11], which is used for regenerating alveolar bone damaged by periodontal disease.
Further, in another aspect, the present invention
[13] A method for treating a subject suffering from periodontal disease, in which a periodontal disease therapeutic agent containing a culture supernatant of mesenchymal cells or mesenchymal stem cells is administered in combination with antibacterial treatment against periodontal pathogens. Concerning treatment methods, including steps.
Further, in another aspect, the present invention
[14] A tooth that is used in the treatment of a subject suffering from periodontal disease and contains a culture supernatant of mesenchymal cells or mesenchymal stem cells and is used in combination with antibacterial treatment against periodontal pathogens. Regarding periodontal disease therapeutic agents.
Further, in another aspect, the present invention
[15] The present invention relates to the use of a culture supernatant of mesenchymal cells or mesenchymal stem cells in the production of a therapeutic agent for periodontal disease used in combination with antibacterial treatment against periodontal pathogens.
Further, in another aspect, the present invention
[16] The present invention relates to a culture supernatant of mesenchymal cells for the treatment of periodontal disease, which is used in combination with antibacterial treatment against periodontal disease pathogens.

According to the periodontal disease therapeutic agent of the present invention, when used in combination with antibacterial treatment, the periodontal disease treatment by scaling and administration of antibiotics, which has been conventionally used as periodontal disease treatment, can be performed in a shorter time. It can produce a high therapeutic effect on periodontal disease.
In particular, according to the periodontal disease therapeutic agent of the present invention, it is possible to promote the regeneration of alveolar bone, which could not be obtained only by periodontal disease treatment such as scaling and administration of antibiotics. According to the periodontal disease therapeutic agent of the present invention, it is possible to promote periodontal tissue regeneration including alveolar bone regeneration in a short period of time even in a patient suffering from severe periodontal disease.
Further, according to the periodontal disease therapeutic agent of the present invention, since the mesenchymal cells themselves are not administered to a living body, problems of safety and quality assurance, which are concerns in cell therapy, can be avoided.

It is an image which shows the oral cavity of the patient before the periodontal disease treatment in Example 5. It is an image which shows the oral cavity of the patient before the periodontal disease treatment in Example 5. FIG. 5 is an image showing the oral cavity of a patient undergoing flap surgery in Example 5. FIG. 5 is an image showing the oral cavity of a patient undergoing flap surgery in Example 5. It is an image which shows the oral cavity of the patient who is administering the culture supernatant containing therapeutic agent after the flap operation in Example 5. FIG. 5 is an image showing the oral cavity of a patient in a state where the incised gingiva is sutured after administration of the culture supernatant-containing therapeutic agent in Example 5. An X-ray image of the oral cavity of a patient on the day (first, third, or fourth) administration of the culture supernatant-containing therapeutic agent in Example 5 is shown. It is a graph which shows the change of the depth of the periodontal pocket in each patient before and after the treatment in the periodontal disease treatment of Example 6. The patient (Mr. S.N.) oral cavity photograph at the time of the first examination and the final evaluation of the periodontal disease treatment of Example 6, and the evaluation of the tooth mobility and the subjective and objective symptoms are shown. The patient (Mr. Y.M.) oral cavity photograph at the time of the first examination and the final evaluation of the periodontal disease treatment of Example 6, and the evaluation of the tooth mobility and the subjective and objective symptoms are shown. The patient (Mr. N.M.) oral cavity photograph at the time of the first examination and the final evaluation of the periodontal disease treatment of Example 6, and the evaluation of the tooth mobility and the subjective and objective symptoms are shown. The patient (Mr. M.Y.) oral cavity photograph at the time of the first examination and the final evaluation of the periodontal disease treatment of Example 6, and the evaluation of the tooth mobility and the subjective and objective symptoms are shown. The patient (Mr. Y.T.) oral cavity photograph at the time of the first examination and the final evaluation of the periodontal disease treatment of Example 6, and the evaluation of the tooth mobility and the subjective and objective symptoms are shown.

As used herein, the term "mesenchymal cell" is a cell that is said to have the ability to differentiate into cells belonging to the mesenchymal system, such as osteoblasts, adipocytes, muscle cells, and chondrocytes. More specifically, dental pulp cells, bone marrow cells, umbilical cord cells, adipocytes and the like can be mentioned. The term "mesenchymal stem cell" means a group of stem cells or progenitor cells capable of differentiating into all or some mesenchymal cells such as osteoblasts, chondroblasts and lipoblasts. More specifically, dental pulp stem cells, bone marrow-derived stem cells, adipose tissue-derived stem cells and the like can be mentioned.

Individuals from which mesenchymal cells or mesenchymal stem cells are derived are of the same species (human origin if human) in order to suppress or avoid rejection in relation to individuals to which this periodontal disease therapeutic agent is applied. ) Is preferably derived, more preferably the same species, and an autologous tissue or autologous cell is used.
In the present invention, the term mesenchymal cell also includes a cell population of mesenchymal cells. At this time, the cell population of mesenchymal cells may include mesenchymal stem cells. That is, the term mesenchymal cell can be read as a cell population of mesenchymal cells or a cell population including mesenchymal cells and mesenchymal stem cells. When a cell population of mesenchymal cells or a cell population containing mesenchymal cells and mesenchymal stem cells is used, the step of selecting mesenchymal stem cells from the cell population is not included, and preparation is performed in this respect. It's simple.
Further, in one embodiment of the present invention, the mesenchymal cells can be replaced with mesenchymal stem cells. That is, as one embodiment, the present invention provides, for example, a periodontal disease therapeutic agent containing a culture supernatant of dental pulp stem cells, which is used in combination with antibacterial treatment against periodontal disease pathogens.

In one preferred embodiment of the invention, the mesenchymal cells are dental pulp cells and the mesenchymal stem cells are dental pulp stem cells. Here, the “dental pulp cell” refers to a dental pulp cell or a cell population thereof recovered / isolated from the dental pulp tissue, and the dental pulp cell obtained by recovering from the dental pulp tissue by enzyme treatment or the like can be used. The dental pulp cells collected from the dental pulp tissue may be cryopreserved and thawed to obtain dental pulp cells. Alternatively, the dental pulp tissue itself may be cryopreserved, thawed, and then subjected to enzyme treatment or the like to be recovered. Preferably, it is an embodiment in which dental pulp cells are used immediately after being recovered from dental pulp tissue by enzyme treatment or the like.
"Pulp stem cells" are included in the pulp cell population recovered from the pulp tissue. Techniques for selecting dental pulp stem cells from dental pulp cells are known (Yamaza et al., "Immunomodulatory properties of stem cells from human exfoliated deciduous teeth". Stem Cell Res Ther. (2010) 1: 5).

In the present specification, "pulp tissue" can be collected from both deciduous teeth and permanent teeth, and can be obtained from the pulp of extracted teeth such as deciduous teeth and wisdom teeth (wisdom teeth) that have been conventionally treated as medical waste. is there. That is, the pulp tissue can be taken out from a tooth extracted dentally in a dental medical facility, or may be taken out from a naturally extracted tooth or a naturally dropped tooth. A method for extracting pulp tissue from a tooth is known, and a person skilled in the art can appropriately carry out the method. If the tooth cannot be frozen immediately on the spot, such as a tooth extracted by dental treatment, the tooth should be placed in a medium such as alpha-minimum essential medium (alpha-MEM) for transportation. It can be soaked and stored and transported at low temperature (for example, 4 ° C).

The origin of the cells (mesenchymal cells, mesenchymal stem cells, dental pulp tissue, dental pulp cells, or dental pulp stem cells) used for preparing the culture supernatant of the present invention is not limited to humans, and other mammals (eg, dental pulp stem cells) Mice, rats, rabbits, dogs, cats, monkeys, sheep, cows, horses).
The cells used to prepare the culture supernatant of the present invention may be derived from permanent teeth or deciduous teeth, but are preferably derived from deciduous teeth that have been shed from the viewpoint of cell proliferation ability. In addition, as long as the obtained culture supernatant is used in combination with antibacterial treatment in the treatment of periodontal disease to achieve an improved treatment effect on periodontal disease and a regeneration effect on periodontal tissue, a large amount of iPS cells, ES cells, etc. Mesenchymal cells (eg, dental pulp cells) or mesenchymal stem cells (eg, dental pulp stem cells) derived from pluripotent stem cells may be used.

A method for collecting dental pulp cells from dental pulp tissue is also known, and for example, it can be carried out according to the method described in Example 1 below. For example, after the dental pulp tissue is shredded using a scalpel or the like, the shredded dental pulp tissue is enzymatically treated with dispase, collagenase, or a mixed enzyme solution thereof. The enzyme treatment depends on the enzyme used, but for example, a mixed enzyme solution in which 4% dispase and 3% collagenase are mixed at a ratio of 1: 1 (when using, the enzyme treatment can be performed at 37 ° C. for 60 minutes. After the treatment, the mixture is sufficiently mixed with a culture solution containing serum, and then impurities are removed using a cell strainer or the like. Then, centrifugation (for example, 2,000 rpm, 4 ° C.) is performed, the supernatant is discarded, and the culture is performed. Dental cells can be collected from the dental pulp tissue by adding the solution. Further, as the enzyme solution, actase (manufactured by Innovative cell technologies; registered trademark) can be preferably used. As described above, the dental pulp A method for collecting dental pulp cells from a tissue is known, and a person skilled in the art can appropriately set the enzyme to be used and test conditions.

The culture supernatant that can be used in the present invention is a culture solution obtained by culturing mesenchymal cells, which is substantially free of cells. Since the periodontal disease therapeutic agent of the present invention is a cell-free culture supernatant, a component effective for periodontal disease treatment is being cultured from mesenchymal cells under certain controlled culture conditions during the culture period. It is preferable because it can be accumulated in.
Further, as a method for separating cell components from the culture solution, a solid-liquid separation method known to those skilled in the art may be applied. Further, it was decided to use a culture supernatant obtained by appropriately subjecting the culture solution to various treatments (for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze-drying, dilution, desalting, storage, etc.). May be good.

For culturing mesenchymal cells, known culturing conditions that are usually performed can be used, and those skilled in the art can appropriately set the conditions for each mesenchymal cell to be used and cultivate.
For culturing mesenchymal cells, a basal medium or a basal medium supplemented with serum or the like can be used. As used herein, the term "basal medium" refers to a medium containing only known components having a low molecular weight, and non-limiting examples of the basal medium include BME (Basal medium Eagle's), MEM (Minimum essential medium), and DMEM (Dulbecco's modified). Eagle's medium), Eagle's medium such as Iskov's modified Dalbeco medium (IMDM), RPMI (Roswell Park Memorial Institue) medium such as RPMI1630, RPMI1640, Fisher's medium, Ham's medium such as F10 medium and F12 medium, MCDB media such as MCDB104, 107, 131, 151, 153, 170, 202 and RITC80-7 medium are known and can be appropriately selected. Two or more kinds of basal media may be used in combination. As an example of the mixed medium, a medium in which IMDM and HamF12 are mixed in equal amounts (for example, commercially available as a trade name: IMDM / HamF12 (GIBCO)) can be mentioned.

Examples of components that can be added to the medium include fetal bovine serum (FBS), human serum, sheep serum, etc., serum replacement (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), and antibiotics. , Various vitamins, various minerals can be mentioned.
Serum and serum substitutes are less than 25% (v / v), less than 20% (v / v), less than 15% (v / v), less than 13% (v / v), 10% of the total medium. It is preferably less than (v / v), less than 7% (v / v), less than 5% (v / v), etc.
As the basal medium used for culturing mesenchymal cells, αMEM containing serum (FBS, human serum, etc.) can be preferably mentioned.

When a periodontal disease therapeutic agent containing a culture supernatant is applied to humans, it is preferable that the culture solution to be collected does not contain animal-derived components (serum or the like) in order to avoid contamination with animal-derived components. For example, the safety can be enhanced by using a medium containing no serum. That is, in one embodiment in the production of the periodontal disease therapeutic agent of the present invention, for example, by culturing the mesenchymal cells in a serum-free medium (serum-free medium), a serum-free culture supernatant can be obtained. Including the step of preparing. It is also preferable that the culture supernatant does not contain antibiotics such as Penicillin-Streptomycin. In one embodiment, an antibiotic-free medium can be used in the final culture for which the culture supernatant is to be collected.
As a serum-free medium, when a periodontal disease therapeutic agent prepared from the collected culture supernatant is used in combination with antibacterial treatment in periodontal disease treatment, the periodontal disease therapeutic effect and periodontal tissue regeneration are improved. It is not limited as long as it is effective. As the serum-free medium, for example, the above-mentioned basal medium or Ringer's solution containing no serum can be used.

Further, for example, when preparing a culture supernatant of mesenchymal stem cells such as dental pulp stem cells, a serum-free medium for stem cells may be used. The serum-free medium for stem cells is a medium capable of maintaining the proliferative property and stem cell characteristics of stem cells, and a commercially available medium can be used. In particular, a serum-free medium for mesenchymal stem cells is preferable. Such serum-free medium for stem cells is not limited to the following, but for example, EXPREP MSC Medium (Biomimetics Sympathies Co., Ltd.), STK2 (registered trademark) Mesenchymal stem cell-free medium (DS Pharma Biomedical Co., Ltd.) ), Mesenchymal Stem Cell Growth Medium DXF (Promo Cell), Stemgro® hMSC Medium (Corning), MesenCult-SF, MesenCult-XF, MesenCult-ACF (STEMCELL Technologies), TheraPEAK MSCGM-CD Mesenchymal Stem Cell Medium, Chemically Defined (Lonza), PRIME-XV® MSC Expansion XSFM, PRIME-XV® MSC Expansion SFM (Irvine Scientific), MSC NutriStem® XF (Biological industries), StemXVivo Xeno-Free Human MSC Expansion Media, StemXVivo Serum-Free Human MSC Expansion Media (R & D Systems), StemMACS MSC Expansion Media kit XF, Human (Miltenyi Biotec), MesenPRO MSC SFM, MesenPRO MSC SFM XenoFree (Gibco), FibroGRO Xeno-Free Human Fibroblast Expansion Medium (Merck) ), PL SOLUTION, PL SOLUTION GMP grade (PL BioScience), etc.

For the culture for preparing the culture supernatant from the mesenchymal cells, it is preferable to carry out one or a plurality of subcultures. In one preferred embodiment, the last or last few subcultures are cultured in serum-free medium. This makes it possible to obtain a culture supernatant containing no serum. On the other hand, serum-free culture supernatant may be obtained by removing serum from the collected culture supernatant by using dialysis, solvent substitution with a column, or the like.
The mesenchymal cells can be cultured under normal temperature and pH conditions, and it is preferable to use an incubator to adjust the culture conditions. Further, the medium is preferably kept so that its composition components can be sufficiently supplied, and can be replaced with a new medium at a frequency of, for example, about once every three or four days.

The step of culturing the mesenchymal cells for preparing the culture supernatant contained in the periodontal disease therapeutic agent of the present invention can include the step of culturing the mesenchymal cells in a medium containing serum. The serum concentration may be contained in the medium within the above range, preferably 10 to 25% (v / v), more preferably about 20% (v / v) with respect to the entire medium. It is a form. Culturing in a medium containing serum is preferably carried out for at least 24 hours or longer, preferably 36 hours or longer, and more preferably 48 hours or longer.
In order to eliminate animal-derived components from the culture supernatant, it is preferable to culture the mesenchymal cells in a serum-free medium for a certain period of time. In one embodiment in the production of the culture supernatant contained in the periodontal disease therapeutic agent of the present invention, the step of culturing the mesenchymal cells in a medium containing serum is followed by further culturing the mesenchymal cells in a serum-free medium. Includes the step of culturing. Culturing in a serum-free medium is preferably carried out for at least 24 hours or longer, preferably 36 hours or longer, and more preferably 48 hours or longer.

Hereinafter, the culture method and the method for collecting the culture supernatant will be illustrated by taking dental pulp cells as an example, but as long as the periodontal disease therapeutic effect and the periodontal tissue regeneration effect are improved when used in combination with the antibacterial treatment in the periodontal disease treatment. Is not limited to the following methods and embodiments. In addition, the following various methods and embodiments can be applied when other cells are used as raw materials.
For example, dental pulp cells are seeded in a culture dish containing the above basal medium and cultured in an incubator adjusted to 5% CO2 and 37 ° C. Subculture is performed as needed. For example, when the cells reach semiconfluent (a state in which cells occupy about 70% of the surface of the culture vessel) or confluence by observing with the naked eye, the cells are separated from the culture vessel and collected, and the culture is filled with the culture solution again. Seed in a container. Subculture may be repeated. For example, subculture is performed 1 to 8 times to grow to the required number of cells (for example, about 1 × 10 ⁶ cells / ml). The cells can be detached from the culture vessel by a conventional method such as trypsin treatment. After the above culture, the cells may be collected and cryopreserved.

Then, the culture supernatant of dental pulp cells is collected. The time of recovery is not limited, and it may be in a semiconfluent state, which is the timing of passage, or it may be recovered at the logarithmic growth stage of cells or at the time of medium exchange. For example, the culture solution can be sucked and collected with a dropper or a pipette. The collected culture supernatant is used as it is or as an active ingredient of the periodontal disease therapeutic agent of the present invention after undergoing one or more treatments. Examples of the treatment here include centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage (for example, 4 ° C., −80 ° C.).

The collected culture supernatant can be appropriately concentrated. That is, the term culture supernatant includes a concentrate of the collected culture supernatant. On the other hand, the culture supernatant can be diluted with an appropriate medium such as a buffer solution. As the concentration method, those skilled in the art can appropriately select and use from known methods. For example, a concentrate of the culture supernatant can be obtained by a spin column concentration method or an ethanol precipitation concentration method. The main culture supernatant may be freeze-dried. That is, the main culture supernatant may be a freeze-dried product.

"Periodontal disease" can be mainly divided into gingival inflammation and periodontitis according to the degree of progression, and occlusal trauma is also known. Periodontitis is an early stage of periodontal disease, and refers to a condition in which the gums are swollen and easily bleed due to plaque or tartar that forms at the root of the tooth or between the teeth. Periodontitis can be broadly classified into mild to moderate to severe according to its symptoms. Mild periodontitis refers to a condition in which the swollen gums are invaded by periodontal disease pathogens and toxins produced by the bacteria and begin to dissolve the alveolar bone. Moderate periodontitis refers to the condition in which the alveolar bone is melted to half the length of the root of the tooth due to periodontal disease. Severe periodontitis refers to a condition in which the alveolar bone is melted by more than half the length of the root of the tooth. When the alveolar bone that supports the tooth is reduced, the tooth sways and eventually falls off. In the present specification, "periodontal disease" also includes peri-implantitis. Peri-implantitis refers to the resorption of alveolar bone that occurs around the implant after implant placement. The periodontal disease therapeutic agent of the present invention also targets the improvement of bone resorption and inflammation (bone thickening and anti-inflammatory) caused by peri-implantitis.
The periodontal disease targeted by the periodontal disease therapeutic agent of the present invention includes the above-mentioned ones, and can particularly target periodontitis and periodontitis caused by periodontal disease pathogens. The present invention is particularly desirable for periodontal disease in which the alveolar bone is absorbed and the periodontal tissue including the alveolar bone needs to be regenerated. The periodontal disease therapeutic agent of the present invention is, in one embodiment, a therapeutic agent for treating periodontal disease in which alveolar bone is absorbed.

The state and degree of progression of periodontal disease can be evaluated by, for example, the depth of the periodontal pocket (PPD), tooth mobility, drainage from the periodontal tissue, bleeding, and the like. The depth of the periodontal pocket can be evaluated by measuring 6 points per tooth (6-point method) using a pocket probe. Tooth mobility can be evaluated based on Miller's criteria. Miller's criteria are 0 degrees (within 0.2 mm of physiological agitation), 1 degree (mild; 0.2 to 1 mm on the lip tongue), 2 degrees (moderate; lip tongue, 1 to 2 mm on the mesio-distal), 3 Classify as degree (altitude; lip tongue, mesio-distal 2 mm or more, or vertical butoh sway). Drainage or bleeding from the periodontal tissue can be assessed by the presence or absence of pus drainage or bleeding. (-) When drainage or bleeding is observed, (+) when drainage or bleeding is observed ((++) when a large amount of drainage or bleeding is observed) .. For specific confirmation of bleeding, insert the pocket probe lightly (with a force of about 15 to 20 g) into the gingival sulcus or pocket, and check the state of bleeding about 20 to 30 seconds after pulling out.
In the present specification, the therapeutic effect of periodontal disease refers to any of the following effects (i) to (iv) and the alveolar bone regeneration effect:
(I) Improvement of periodontal pocket depth (PPD) (ii) Improvement of tooth mobility (iii) Improvement of drainage from periodontal tissue (iv) Improvement of bleeding from periodontal tissue It can be confirmed by the evaluation by the above-mentioned method. Further, in the present specification, the regeneration of the periodontal tissue including the alveolar bone is defined as the periodontal bone absorbed by the periodontal disease before and after the administration of the periodontal disease therapeutic agent of the present invention. The state of recovery to approach the state of. Although it depends on the condition of the periodontal tissue to be treated, the periodontal disease therapeutic agent of the present invention has all the effects of the above-mentioned effects (i) to (iv) and the alveolar bone regeneration effect in one preferable embodiment. Play.

The "periodontal disease therapeutic agent" of the present invention contains a culture supernatant of mesenchymal cells and is used in combination with antibacterial treatment in periodontal disease treatment. In a preferred embodiment, the periodontal disease therapeutic agent comprises a culture supernatant of dental pulp cells.
In addition, the periodontal disease therapeutic agent of the present invention may contain a bioabsorbable material, a gelling agent, and other pharmaceutically acceptable components. Examples of the bioabsorbable material include hyaluronic acid, collagen, fibrinogen and the like as the organic bioabsorbable material. Further, as the gelling material, it is preferable to use a material having high biocompatibility, and hyaluronic acid, collagen, fibrin glue and the like can be used. Various types of hyaluronic acid and collagen can be selected and used, but are preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.). Other components permitted in the formulation include, for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, saline and the like. be able to. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethyl cellulose, calcium carbonate and the like can be used. As the buffer, phosphate, citrate, acetate and the like can be used. As the emulsifier, gum arabic, sodium alginate, tragant and the like can be used. As the suspending agent, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the pain-relieving agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. Propylene glycol, ascorbic acid and the like can be used as the stabilizer. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like can be used. As the preservative, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used. It may contain antibiotics, pH regulators, growth factors (eg, epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF)) and the like.

The dosage of the periodontal disease therapeutic agent containing the culture supernatant of the mesenchymal cells of the present invention has improved periodontal disease therapeutic effect and periodontal tissue regeneration when used in combination with antibacterial treatment for periodontal disease treatment. It is not particularly limited as long as the effect can be obtained. A person skilled in the art can set it in consideration of the age, weight, pathological condition, etc. of the test subject. Although not limited to the following, the administration schedule may be, for example, once every 3 to 5 days, once every 1 to 2 weeks, once every 1 to February, or the like. In preparing the administration schedule, the gender, age, body weight, pathological condition, etc. of the subject (recipient) can be taken into consideration.

The dose depends on the condition of the tooth and periodontal tissue suffering from periodontal disease, the administration route, the age of the patient, or the body weight. As long as it has a therapeutic effect, it is not limited to the following, but for example, when the culture supernatant collected after culturing at a cell density in a semiconfluent state is used as it is for the treatment of periodontal disease, when it is administered to an adult as an active ingredient amount, for example, It can be administered in the range of 0.1 to 10 ml / dose per tooth, and preferably in the range of 0.1 to 1 ml / dose. This may be administered once or several times.

The administration route of the periodontal disease therapeutic agent of the present invention is oral administration, and it is preferable to administer it directly to the tooth and / or periodontal tissue suffering from periodontal disease. Methods of administration include injection, application or spraying into the affected area. The form of the periodontal disease therapeutic agent of the present invention is not particularly limited, but a formulation form such as a liquid, powder, pellet, cream, or granule suitable for administration to the affected area is preferable. The periodontal disease therapeutic agent of the present invention is preferably administered directly to the affected area by the combined use of flap surgery or the like, but can also be administered to the periodontal tissue and periodontal pocket without the combined use of flap surgery or the like. The first dose in the treatment regimen is preferably combined with flap surgery.

Examples of the target individual to which the periodontal disease therapeutic agent of the present invention is applied include mammals including humans (pets, livestock, laboratory animals, etc.). For example, in addition to humans, dogs, cats, rabbits, mice, cows, pigs, goats, sheep, horses, monkeys, guinea pigs, rats and mice can be mentioned.

As used herein, the term "antibacterial treatment" is an antibacterial treatment that can be used for periodontal disease treatment together with a periodontal disease therapeutic agent containing a culture supernatant of mesenchymal cells, and is a periodontal disease that causes periodontal disease. Treatment performed to remove pathogens or their derived plaque, tartar, biofilm, etc. Not limited to, for example, administration of antibacterial agents, administration of disinfectants such as chlorhexidine, laser irradiation such as carbon dioxide laser, periowave, erbium yag (Er: YAG) laser, brushing and scaling, root planing, teeth Includes cleaning inside the peripheral pocket. Antibacterial treatment can also be performed by combining multiple antibacterial treatments, such as a combination of root planing and administration of an antibacterial agent. In one preferred embodiment of the invention, the antibacterial treatment is administration of an antibacterial agent. Also in a more preferred embodiment, the antibacterial treatment comprises scaling and / or root planing and administration of an antibacterial agent.

The "antibacterial agent" that can be used in the present invention is not limited as long as it is an antibiotic or an antibacterial agent having an antibacterial action against periodontal disease pathogens, and examples thereof include tetracycline antibiotics and macrolide antibiotics. For example, minocycline hydrochloride, tetracycline, doxycycline, erythromycin, clarithromycin, loxythromycin, azithromycin and the like can be mentioned. Examples of the antibacterial agent include quaternary ammonium salts such as cetylpyridinium chloride, bisguanides such as chlorhexidine, fluoroquinolone antibacterial agents such as sparfloxacin and levofloxacin, and triclosan. More specifically, the tetracycline antibiotic is a composition for treating periodontal disease containing minocycline or a pharmaceutically acceptable salt, hydrate, or solvate thereof (Japanese Patent Laid-Open No. 2001-335477). , Japanese Patent Application Laid-Open No. 11-286448, Japanese Patent Publication No. 1-12728, Japanese Patent Application Laid-Open No. 2-3425, etc.). In a preferred embodiment, the antibacterial agent comprises minocycline or a pharmaceutically acceptable salt thereof. As an antibacterial agent containing minocycline or a pharmaceutically acceptable salt thereof, commercially available "Perioclin Dental Ointment" (sold by Sunstar Inc.) or "Periofeel Dental Ointment" (sold by Showa Yakuhin Kako Co., Ltd.) is used. You may.

The type of physiologically acceptable salt of minocycline is not particularly limited, and for example, mineral acid salts such as hydrochlorides, sulfates and acetates, and organic acid salts such as methanesulfonates can be used. Minocycline or its physiologically acceptable salt may be a hydrate or a solvate. The solvent for forming the solvate is not particularly limited as long as it is physiologically acceptable, but a solvate such as ethanol can be used. It is preferable to use hydrochloride as the salt of minocycline. The content of minocycline or a physiologically acceptable salt thereof contained in the pharmaceutical composition of the present invention is generally about 0.01 to 15% by weight (free form equivalent amount) with respect to the weight of the composition. Yes, preferably about 0.1 to 5% by weight. Further, as other components, an oily base, a buffer; a pH adjuster; a surfactant; a plasticizer; a binder; a dispersant; a preservative; and / or a colorant may be appropriately added to the composition. Good.

It is desirable that the antibacterial agent of the present invention is provided, for example, as a viscous liquid or paste-like composition, preferably suitable for topical administration to the oral cavity or periodontal tissue. However, the dosage form is not limited to liquid or paste, and may be a dosage form suitable for oral administration as long as it is effective as an antibacterial agent in the treatment of periodontal disease.
The periodontal disease therapeutic agent containing the antibiotic of the present invention can be administered to a patient by directly applying it directly to an affected area (for example, a periodontal disease area), preferably a periodontal pocket, in more detail. Is locally administered to the damaged area of the periodontal tissue. The amount of application can be appropriately selected according to the size of the affected area, the degree of the disease, and the like. For example, about 1 to 100 mg of a periodontal disease therapeutic agent containing minocycline or a pharmaceutically acceptable salt thereof in an amount of about 0.01 to 15% by weight (free form equivalent amount) is administered at a rate of about 1 to 100 mg per tooth, preferably 1 to 10 mg. can do. When used in combination with the periodontal disease therapeutic agent of the present invention, the periodontal disease therapeutic effect can be obtained even if the amount of minocycline or a pharmaceutically acceptable salt used is suppressed. In one embodiment, the amount of minocycline or pharmaceutically acceptable salt used in a single treatment is less than 10 mg / dose (1 mg or more and less than 10 mg, more preferably 1 mg or more and 5 mg or less, still more preferably 1 mg). It can be 2 mg or less). This can reduce the dose of minocycline or a pharmaceutically acceptable salt. The number of administrations and the administration period can be appropriately selected, but in a preferred embodiment in which the dose can be suppressed, the number of administrations is once every two weeks, once every three weeks, or one month. It can be administered once every time.

In the present specification, "combining" a periodontal disease therapeutic agent containing a culture supernatant and an antibacterial treatment against a periodontal disease pathogen means that the timing of administration of the periodontal disease therapeutic agent and the timing of the antibacterial treatment are simultaneously performed, or Includes doing before and after at regular intervals in chronological order.
When administration and treatment are performed at the same time, it can be repeated multiple times (for example, 2, 3, 4, 5 or more) at regular intervals depending on the course of treatment. The intervals are, for example, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week. , 2 weeks or 3 weeks.
When the periodontal disease therapeutic agent and antibacterial treatment are sequentially administered, the administration or treatment is performed at regular intervals. The intervals are, for example, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week. , 2 weeks or 3 weeks. The administration of the periodontal disease therapeutic agent and the antibacterial treatment may be alternately performed. Antibacterial treatment is first performed once or multiple times (eg, 2, 3, 4, 5 times or more), and then the periodontal disease therapeutic agent is administered once or multiple times (eg, twice). , 3, 4, 5 or more) may be administered. In addition, the administration of the periodontal disease therapeutic agent and the simultaneous combination and the sequential combination of the antibacterial treatment may be combined alternately or randomly according to the symptom of the target.
A preferred embodiment of the present invention is a form in which administration of a periodontal disease therapeutic agent and antibacterial treatment are performed at the same time.
The periodontal disease therapeutic agent of the present invention is shorter with a smaller amount of antibacterial agent than the periodontal disease treatment by scaling and administration of antibacterial agent, which has been conventionally used as periodontal disease treatment when used in combination with antibacterial treatment. It is possible to achieve a high therapeutic effect on periodontal disease in time. This makes it possible to suppress the occurrence of side effects and resistant bacteria, which have been problems in the administration of antibiotics in the past.
When the administration of the periodontal disease therapeutic agent and the antibacterial treatment are performed at the same time, the present invention is not limited to the following forms, but for example, in one embodiment, a periodontal disease therapeutic agent in which the culture supernatant and the antibacterial agent are mixed in advance is prepared. Then, the form of applying directly to the affected area can be mentioned. Such a periodontal disease therapeutic agent may be prepared so that the culture supernatant and the antibacterial material are contained within the above dose range.
On the other hand, the case where the administration of the periodontal disease therapeutic agent and the antibacterial treatment are carried out before and after is not limited to the following forms, but the periodontal disease therapeutic agent containing the culture supernatant is directly administered to the affected area. Then, an antibacterial agent may be separately orally administered.

The periodontal disease therapeutic agent of the present invention is used in combination with a therapeutic method for the purpose of periodontal tissue regeneration such as GTR method, bone graft material, collagen sponge, or b-FGF in one embodiment. Can be done.
As used herein, the term "bone graft material" refers to autologous bone or artificial bone that can be used in combination for regeneration of periodontal tissue damaged by periodontal disease, such as hydroxyapatite (HA) and tricalcium phosphate. It refers to artificial bone whose constituents are calcium (TCP), β-calcium phosphate (β-TCP) activated glass, and the like. In addition, recombinant human b-FGF (basic fibroblast growth factor) may also be administered in combination, and a commercially available ligros dental solution set (Kaken Pharmaceutical Co., Ltd .; periodontal tissue regeneration agent and recombinant human b) -FGF (including basic fibroblast growth factor)) can be used in combination.
These therapeutic methods for the purpose of regenerating periodontal tissue can also be used for treatment when a therapeutically effective amount of the periodontal disease therapeutic agent of the present invention is administered to the affected area. Although not limited to the following, for example, after applying the periodontal disease therapeutic agent of the present invention and antibacterial treatment to the affected area, the membrane, bone graft material, and collagen sponge used in the GTR method can be applied to the affected area.

Hereinafter, the present invention will be described based on examples, but the following examples are provided for purposes of illustration only. Therefore, the scope of the present invention is not limited to the above-described embodiment or the following embodiment, but is limited only by the appended claims. In addition, the contents of the literature disclosed herein are incorporated herein by reference.

(Example 1: Collection of dental pulp cells from dental pulp tissue)
1. 1. Reagents ・ α-MEM medium (containing 20% FBS and 1% Penicillin-Streptomycin)

2. Collection of pulp tissue and pulp cells from extracted deciduous teeth All of the following operations were performed in a safety cabinet in the Cell Processing Center (CPC).
Pulp tissue was removed from the extracted milk teeth collected from a patient (4 to 13 years old) at a partner dental clinic using tweezers or the like. The dental pulp tissue was cut into small pieces using a scalpel or the like, and treated with an enzyme solution for tissue dispersion such as Accutase (registered trademark) at 37 ° C. for 30 minutes to disperse the cells. The dispersed cells were suspended in α-MEM medium containing 20% FBS, and the number of cells was counted. Based on the result of counting the number of cells, the cells were cultured using a cell culture flask of an appropriate size (37 ° C, 5% CO ₂ ).

(Example 2. Subculture and cryopreservation)
1. 1. Reagent (Example 2 and below, similar reagents were used)
-Α-MEM medium (containing 20% FBS and 1% Penicillin-Streptomycin)
・ TrypLE Select (gibco / 12604-013)
・ Cerbanker 2 (Nippon Zenyaku Kogyo)

2. Expanded culture The primary cultured cells collected in the flask in Example 1 above were cultured in the flask until they reached a semiconfluent (cell density: 70 to 80%) state. It was confirmed by microscopic examination that it was in a semiconfluent state, and the entire amount of the medium was removed. The bottom of the flask was washed with an appropriate amount of D-PBS (-) (gibco / 14190-144) in order to completely remove the medium component containing serum, and this operation was repeated twice. After washing, an appropriate amount of TrypLE Select (gibco / 12563-011) was added, spread over the entire bottom surface of the flask, and incubated at 37 ° C. When some cells were detached from the bottom of the flask, the liquid was slowly circulated in the flask to detach the cells from the bottom of the flask. The flask was tilted and α-MEM medium was added so as to flow through the entire flask, and the cells were peeled off to prepare a cell suspension. The cell suspension was collected in a 15 mL tube. A new α-MEM medium was added so as to flow through the entire flask, and the cells were peeled off. The cell suspension was collected in a 15 ml tube. It was confirmed by microscopic examination that the cells were detached from the flask. The collected cell suspension was centrifuged, the supernatant was removed, α-MEM medium was added and suspended, and the number of cells was counted. Culturing was performed in an incubator at 37 ° C. and 5% CO ₂ using a cell culture flask having a cell density of 2,000 to 5,000 cells / cm ^{2 and an appropriate size.} Expanded culture was performed using α-MEM medium, and cryopreservation was performed.

3. 3. Cryopreservation After confirming that the cells in the expanded culture were in a semiconfluent (cell density: 70-80%) state, the culture supernatant was collected in a new 50 mL tube. The cells in the flask after collecting the supernatant were frozen, and the culture supernatant collected in a 50 mL tube was used for various tests. HBV quantification, HCV quantification, HIV quantification, parvo IgG, parvo IgM, CMV-IgG, CMV-IgM, FTA-ABS, STD-mycoplasma identification, cells suitable for use in periodontal disease treatment by tests including endotoxin. It was confirmed.

The bottom of the flask was washed with D-PBS (-) in order to completely remove the medium components including serum from the flask after the culture supernatant was collected and became semi-confluent during the expansion culture. Was repeated twice for each flask. TrypLE Select was added to each flask, spread over the entire bottom surface of the flask, and incubated at 37 ° C. When some cells were detached from the bottom of the flask, the liquid was slowly circulated in the flask to detach the cells from the bottom of the flask. α-MEM medium was added so as to flow through the entire flask, and the cells were peeled off to prepare a cell suspension. The cell suspension was collected in 15 mL tubes. 3 mL of α-MEM medium was newly added to each flask so as to flow through the entire flask, and the remaining cells in the flask were peeled off to form a cell suspension, which was collected in a 15 ml tube. The collected cell suspension was centrifuged, then the supernatant was removed, D-PBS (-) was added to suspend the suspension, and the number of cells was counted. Centrifugation was performed again to remove the supernatant. Cerbunker 2, which is a cryopreservation solution in an amount corresponding to the number of cells (final concentration: 0.9 to 1.3 × 10 ⁶ cells / ml), was added and gently pipetting was performed. The cryopreservation solution containing the cells was transferred to a cryotube in an amount of 1 mL each. The cryotube was placed in a bicelle, frozen in a -80 ° C freezer, and transferred to a liquid nitrogen tank for storage within 3 days.

(Example 3: Preparation of dental pulp cells for collection of culture supernatant)
2. Raise cells Frozen extracellular fluid containing cryopreserved dental pulp stem cells was rapidly thawed in a constant temperature bath for about 70%. About 70% of the thawed frozen cell solution was added to a medium (αMEM medium) heated to 37 ° C. After thawing, centrifugation was performed, and then the supernatant was removed. A new medium was added and the number of cells was counted. Culturing was performed in αMEM medium in an incubator at 37 ° C. and 5% CO ₂ using a cell culture flask having a cell density of 2,000 to 5,000 cells / cm ^{2 and an appropriate size.} After sowing, the whole amount of medium was changed every 2 to 3 days.

3. 3. Cells were cultured in a flask until the cells were semi-confluent (cell density: 70-80%). It was confirmed by microscopic examination that it was in a semiconfluent state, and the entire amount of the medium was removed. The bottom of the flask was washed with an appropriate amount of D-PBS (-) (gibco / 14190-144) in order to completely remove the medium component containing serum, and this operation was repeated twice. After washing, an appropriate amount of TrypLE Select (gibco / 12563-011) was added, spread over the entire bottom surface of the flask, and incubated at 37 ° C. When some cells were detached from the bottom of the flask, the liquid was slowly circulated in the flask to detach the cells from the bottom of the flask. The flask was tilted and α-MEM medium was added so as to flow through the entire flask, and the cells were peeled off to prepare a cell suspension. The cell suspension was collected in a 15 mL tube. A new α-MEM medium was added so as to flow through the entire flask, and the cells were peeled off. The cell suspension was collected in a 15 ml tube. It was confirmed by microscopic examination that the cells were detached from the flask. The collected cell suspension was centrifuged, the supernatant was removed, α-MEM medium was added and suspended, and the number of cells was counted. Culturing was performed in α-MEM medium in an incubator at 37 ° C. and 5% CO ₂ using a cell culture flask having a cell density of 2,000 to 5,000 cells / cm ^{2 and an appropriate size.}

(Example 4: Recovery of culture supernatant)
The dental pulp cells after the subculture of Example 3 were cultured in a flask containing αMEM medium containing no serum. The culture was carried out at 37 ° C. and 5% CO ₂ for 72 hours. Then, the medium used for culturing was collected in a new 15 mL tube without discarding. The recovered medium was centrifuged. After centrifugation, only the supernatant (not including the sediment) was collected in a new 15 mL tube and used as a culture supernatant derived from dental pulp cells.

(Example 5: Periodontal disease treatment with combined use of culture supernatant and minocycline 1)
A male patient (49 years old) suffering from periodontal disease was treated with a culture supernatant of dental pulp stem cells and minocycline.
The chief complaint from the male patient was pain associated with agitation and bleeding in the upper left second tooth. Photographs of the oral cavity before treatment are shown in FIGS. 1 and 2. The clinical findings of the male patient were gingival redness and swelling and occlusal pain. The probing depth (PPD) in the upper left second periodontal pocket was 10 mm, the agitation was 3 degrees (2 mm or more), pus drainage (++), and bleeding BOP (++). An X-ray examination revealed a vertical bone resorption image of the upper left second centrifuge.
Initial treatment for periodontal disease was started in June 2017. Specifically, after the Tooth Brushing Instruction (TBI), scaling route planing was performed to improve anti-inflammatory and oral hygiene status.
On August 4, 2017, flap surgery (incision of the gingiva, removal of defective granulation tissue and scaling root planing of the root surface) was performed on the upper left second tooth and gingiva (intraoral photograph during flap surgery). 3 and 4). At that time, a therapeutic agent containing the culture supernatant of dental pulp stem cells and minocycline (therapeutic agent containing the culture supernatant) prepared in Example 4 was applied to the root surface and the bone defect site of the alveolar bone. Specifically, 0.25 ml of the culture supernatant and 0.1 g of Periofeel (registered trademark) dental ointment 2% (Showa Yakuhin Kako Co., Ltd .; containing 10 mg (titer) of minocycline hydrochloride in 0.5 g) are injected. A therapeutic agent containing a culture supernatant was prepared by adding to a syringe (disposable / nipro syringe and niproblunt needle) and mixing until mechanically gelled using a sterilized metal stirring rod. Then, the root surface was surface-treated with EDTA, and the entire amount of the therapeutic agent containing the culture supernatant was applied to the root surface and the bone defect site of the alveolar bone with the injection syringe (FIG. 5). After application, the incised gingiva was closed and sutured (Fig. 6).
Then, as described in the treatment history below, the therapeutic agent containing the culture supernatant was applied three times. The second and subsequent applications of the culture supernatant-containing therapeutic agent were applied into the periodontal tissue and the periodontal pocket without using flap surgery.

The clinical findings of the fourth treatment with the culture supernatant-containing therapeutic agent (85 days after the first treatment with the culture supernatant-containing therapeutic agent) were that the periodontal pocket PPD was 4 mm and the agitation was It was 1 degree (0.2 mm or less), drainage (-), and bleeding BOP (-). In addition, as a result of the X-ray examination, it was possible to observe the regeneration of the alveolar bone absorbed by periodontal disease (Fig. 7).

各患者の「自覚症状」、「他覚症状」、「歯周ポケットの深さ」、「増骨」について下記表のスケジュールに従い評価した。また、前記４つの項目に加えて、「歯の動揺度数」について試験開始前と最終評価時に測定・評価した。

（最終評価は、各患者の治療終了時点または治療から一定期間経過後に行った。具体的には、S.N氏は2017年12月4日、Y.M氏は2017年12月11日、N.M氏は2018年2月16日、M.Y氏は2018年3月16日、Y.T氏は2018年4月4日に最終評価を行った）(Example 6: Periodontal disease treatment by combined use of culture supernatant and minocycline 2)
Five patients (20-85 years old) suffering from periodontal disease were treated with a culture supernatant of dental pulp stem cells and minocycline. The culture supernatant of dental pulp stem cells was prepared in Example 4 above, and the same minocycline as in Example 5 was used. The table below shows the ages of the five patients, periodontal pockets before the start of the study, the degree of agitation, and the presence or absence of subjective and objective symptoms.

The "subjective symptoms", "objective symptoms", "periodontal pocket depth", and "bone augmentation" of each patient were evaluated according to the schedule in the table below. In addition to the above four items, "tooth mobility" was measured and evaluated before the start of the test and at the final evaluation.

(The final evaluation was performed at the end of each patient's treatment or after a certain period of time from the treatment. Specifically, Mr. SN was on December 4, 2017, Mr. YM was on December 11, 2017, and Mr. NM was in 2018. February 16, 2018, MY made the final evaluation on March 16, 2018, YT made the final evaluation on April 4, 2018)

The table below shows the dose, frequency of administration, and duration of administration of the culture supernatant and minocycline for each patient. In the table, FOP was performed to indicate that flap surgery was performed.

5 The results of changes in the periodontal pocket before and after treatment in each patient are shown in FIG. In addition, FIGS. 9 to 13 show a photograph of the oral cavity, the degree of agitation, and evaluation of other subjective symptoms at the time of the first examination and the final evaluation of each patient.
As shown in FIG. 8, the depth of the periodontal pocket was reduced by an average of about 25% in each patient after treatment. Further, as shown in FIGS. 9 to 13, the tooth mobility improved in all patients, and the average tooth mobility decreased from 2.6 to 1.0. In addition, all patients had occlusal pain, dull pain, drainage, and bleeding at the first visit, but occlusal pain, dull pain, drainage, and bleeding disappeared in all patients at the final evaluation.

Periodontal disease treatment with conventional scaling root planing is a procedure that reduces bacteria and their metabolites. In addition, subgingival scaling and root planing performed using a curette type scaler is performed on the subgingival area, that is, the tooth surface facing the periodontal pocket, and the operation becomes complicated as the periodontal pocket becomes deeper. It requires appropriate skill, time and effort.
For pocket curettage, if there is no improvement in scaling root planing, the bacterial plaque and tartar on the root surface facing the periodontal pocket are removed, and then the cementum on the root surface that is further contaminated is removed. At the same time, the periodontal pocket epithelial layer including the junctional epithelium of the inner wall of the periodontal pocket and the inflammatory connective tissue are scraped using a curette type scaler. It is expected that periodontal pocket curettage will improve the inflammation of the periodontal tissue and make the periodontal pocket shallower.
As described above, in the conventional scaling, root planing, and periodontal disease treatment by pocket curettage, the main objectives are to improve the inflammation of the periodontal tissue and to make the periodontal pocket shallow. Similarly, administration of antibiotics such as periofeel also aims to reduce the total number of periodontal pathogenic bacteria in the periodontal pocket, and therefore, as shown in Example 5 of this time, generally alveolar bone. Can not be expected to regenerate.
The total amount of minocycline hydrochloride used for treatment in Example 5 was about 8 mg after four administrations. Compared with the conventional dose of dental ointment containing minocycline hydrochloride (using 10 mg (titer) of minocycline hydrochloride once a week), a small amount of administration was effective in treating periodontal disease.

Claims (12)

A periodontal disease therapeutic agent containing a culture supernatant of mesenchymal cells, which is used in combination with antibacterial treatment against periodontal disease pathogens. The periodontal disease therapeutic agent according to claim 1, wherein the antibacterial treatment is selected from the group consisting of administration of an antibacterial agent, administration of a disinfectant solution, laser irradiation, scaling, root planing, and periodontal pocket cleaning. A treatment for periodontal disease, which is one of the treatments. The periodontal disease therapeutic agent according to claim 1 or 2.
A periodontal disease therapeutic agent, wherein the antibacterial treatment is administration of a periodontal disease therapeutic agent containing minocycline or a pharmaceutically acceptable salt thereof. The periodontal disease therapeutic agent according to claim 3.
A therapeutic agent for periodontal disease used at the same time as administration of minocycline or a pharmaceutically acceptable salt thereof. The periodontal disease therapeutic agent according to claim 3 or 4.
A periodontal disease therapeutic agent in which the amount of minocycline or a pharmaceutically acceptable salt thereof administered in combination with the periodontal disease therapeutic agent is less than 10 mg at a time. The periodontal disease therapeutic agent according to any one of claims 1 to 5.
A therapeutic agent for periodontal disease, wherein the culture supernatant of the mesenchymal cells is a culture supernatant obtained by culturing a mesenchymal cell group containing mesenchymal stem cells. The periodontal disease therapeutic agent according to claim 6.
A therapeutic agent for periodontal disease, wherein the mesenchymal cells are dental pulp cells and the mesenchymal stem cells are dental pulp stem cells. The periodontal disease therapeutic agent according to any one of claims 1 to 7.
A periodontal disease therapeutic agent used in combination with at least one periodontal disease treatment selected from the group consisting of GTR method, β-TCP, bone graft material, collagen sponge, and b-FGF. The periodontal disease therapeutic agent according to any one of claims 1 to 8.
A periodontal disease therapeutic agent for application to periodontal tissue inflamed or damaged by periodontal disease. The periodontal disease therapeutic agent according to any one of claims 1 to 9.
A periodontal disease therapeutic agent, wherein the culture supernatant of the mesenchymal cells is a culture supernatant obtained by culturing the mesenchymal cells using a serum-free medium. The periodontal disease therapeutic agent according to claim 10.
The culture supernatant of the mesenchymal cells is a culture supernatant obtained by culturing the mesenchymal cells using a medium containing serum and subsequently culturing the mesenchymal cells using a serum-free medium. Peri-illness therapeutic agent. The periodontal disease therapeutic agent according to any one of claims 1 to 11, which is used for regenerating alveolar bone damaged by periodontal disease.

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