JPWO2010052814A1 - Hard tissue regeneration treatment composition - Google Patents

Abstract

[Summary] mutans and / or P. mutans. A composition for treating hard tissue regeneration comprising gingivalis is provided. SOLUTION: S. mutans and / or P. mutans. By including a low concentration of gingivalis, it was possible to provide a composition for treating hard tissue regeneration. [Selection figure] None

Description

  The present invention relates to S.I. mutans (Streptococcus mutans) and / or P. mutans. The present invention relates to a composition for treating hard tissue regeneration comprising gingivalis (Porphyromonus gingivalis). More specifically, the present invention relates to the hard tissue regeneration treatment composition contained as an active ingredient in a therapeutic agent for regeneration of dentin and / or periodontal tissue and bone tissue.

  Conventionally, S.M. mutans is a causative agent of caries (Caries), P. pneumoniae. gingivalis has been the causative agent of periodontal disease. Therefore, for the treatment and prevention of caries and periodontal disease, S. A basic antibacterial peptide having a bactericidal activity against mutans and having a cell proliferating effect on mesenchymal stem cells (see, for example, Patent Document 1), and P. coli containing polyphenol as an active ingredient. gingivalis adhesion inhibitor to periodontal tissue (see, for example, Patent Document 2), P. gingivalis. Immunogenic polypeptides containing specific amino acids for treating or preventing diseases and infections caused by gingivalis (see, for example, Patent Document 3) have been developed.

However, S. mutans and P.M. gingivalis is a bacterium that is resident in the oral cavity and does not necessarily cause caries or periodontal disease, but low-level infection of these bacteria can prevent caries and periodontal disease. It was thought to be triggered.
In the clinical field, it has been observed that dentin regenerates at the stage where caries is induced, contrary to the progress of caries. This is thought to be caused by the dentin being melted by caries and the growth factors present in the dentin matrix stimulating the odontoblasts through the dentinal tubules.
Therefore, the present inventors may activate the proliferation of cells such as dental pulp cells and periodontal ligament cells due to the infection with a low concentration of bacteria, which may promote the regeneration of dentin and periodontal tissue. Therefore, the present inventors examined a composition for treating hard tissue regeneration containing these bacteria.

As dentine and periodontal tissue regenerating agents, pulp capping agents with calcium hydroxide and periodontal tissue regenerating agents with growth factors are provided, but it is difficult to achieve a 100% success rate in either case However, there are still points to be improved. Accordingly, it is expected that a new composition for treating hard tissue regeneration will be provided.
[Prior documents]
JP 2005-281225 A Japanese Patent No. 3837172 Special table 2007-529195 gazette

  The present invention relates to S.I. mutans and / or P. mutans. It is an object of the present invention to provide a composition for treating hard tissue regeneration containing gingivalis.

  As a result of intensive studies to solve the above problems, the present inventors have found that mutans and / or P. mutans. When gingivalis is present at a high concentration, it acts as a causative agent of caries and periodontal disease, but when it is present at a low concentration, it promotes differentiation of dental pulp cells into odontoblasts or osteoblasts. I found out. Therefore, S.M. mutans and / or P. mutans. A composition for treating hard tissue regeneration containing a low concentration of gingivalis was obtained, and the present invention was completed.

That is, the present invention relates to the following hard tissue regeneration treatment compositions (1) to (8).
(1) S.M. mutans and / or P. mutans. A composition for treating hard tissue regeneration comprising gingivalis.
(2) S.M. mutans and / or P. mutans. The composition for hard tissue regeneration treatment according to the above (1), wherein gingivalis is a crushed cell.
(3) S.M. mutans and / or P. mutans. The composition for hard tissue regeneration therapy according to the above (1) or (2), wherein the concentration of the protein contained in the disrupted gingivalis cell is less than 100 μg / ml.
(4) The composition for hard tissue regeneration treatment according to any one of the above (1) to (3), which is used for regeneration of dentin and / or periodontal tissue.
(5) A pulp capping agent containing the composition for treating hard tissue regeneration according to any one of (1) to (4) as an active ingredient.
(6) A therapeutic agent for bone tissue regeneration comprising the composition for treating hard tissue regeneration according to any one of (1) to (4) as an active ingredient.
(7) S.M. mutans and / or P. mutans. A composition for promoting cell differentiation, comprising gingivalis.
(8) S.M. mutans and / or P. mutans. A composition for improving alkaline phosphatase (hereinafter referred to as ALP) activity of cells containing gingivalis.

  By regenerating dentin and / or periodontal tissue with the composition for the treatment of hard tissue regeneration of the present invention, S. cerevisiae such as caries and periodontal disease can be obtained. mutans and / or P. mutans. It becomes possible to treat or prevent diseases and infections caused by gingivalis.

S. It is the figure which showed the ALP activity of the composition for hard tissue regeneration treatment containing mutans (Test Example 1). P. It is the figure which showed the ALP activity of the composition for hard tissue regeneration treatment containing gingivalis (Test Example 1). It is the figure which showed BSP expression of hDPF by the composition for hard tissue regeneration treatment (Test Example 1).

  The “composition for regenerating and treating hard tissue” of the present invention comprises S. mutans, P.M. It is a composition containing gingivalis or a combination thereof, and any composition can be used as long as it can be used for regeneration of hard tissues such as bones, teeth, hair, and nails. It is particularly preferable that it can be used for regeneration of dentin, periodontal tissue and the like.

  S. cerevisiae contained in the “hard tissue regeneration treatment composition” of the present invention. mutans, P.M. As long as it is safe when administered to the human body or the like as a “hard tissue regeneration treatment composition”, gingivalis is any one such as one stored in a bacterial depository or one that is commercially available. Can be used. In addition, patient-derived S.P. mutans, P.M. Gingivalis can also be collected and included per se.

  These bacteria included in the “composition for hard tissue regeneration treatment” of the present invention may be live bacteria or killed killed bacteria as long as they are effective as the “hard tissue regeneration treatment composition”. However, dead bacteria are more preferable in terms of safety.

  These bacteria contained in the “composition for regenerating and treating hard tissue” of the present invention are preferably contained so that the concentration of the protein contained in the disrupted microbial cells is less than 100 μg / ml, particularly 10 μg / ml or less. It is preferable that it is contained at a low concentration.

  The “composition for regenerating and treating hard tissue” of the present invention is preferably one that can be used for regenerating hard tissues such as bones, teeth, hair, and nails, and particularly acts on dental pulp cells and periodontal ligament cells. It is preferable to be able to be used for regeneration of dentin, periodontal tissue, or any of these.

The “composition for regenerating and treating hard tissue” of the present invention can be used as an active ingredient of a pulp capping agent or a therapeutic agent for regenerating bone tissue.
Examples of the “dental pulp capping agent” include those using the “composition for regenerating and treating hard tissue” of the present invention as it is or those combined with calcium hydroxide or the like. Examples of the “treatment agent for bone tissue regeneration” include those using the “hard tissue regeneration treatment composition” of the present invention as they are, or those combined with growth factors such as collagen, BMP and FGF. It is done. This “therapeutic agent for bone tissue regeneration” is a method of injecting into a bone defect part with a syringe in order to regenerate the bone tissue, or by soaking it in a collagen sponge or the like. It can be used for a transplanting method and the like.
Furthermore, the “composition for regenerating and treating hard tissue” of the present invention can be used as a periodontal tissue regenerating agent in combination with a growth factor or the like.
These “dental pulp capping agents” and “therapeutic agents for bone tissue regeneration” are not particularly limited in the form of drugs, but in combination with generally known gels, moisturizers, thickeners, etc. It can also be provided as an agent or a coating agent.

  The “composition for promoting differentiation” of the present invention refers to S.I. mutans and / or P. mutans. It includes gingivalis and includes any one that induces or promotes cell differentiation. For example, S. cerevisiae that promotes differentiation of dental pulp cells into odontoblasts or osteoblasts. mutans and / or P. mutans. A composition containing gingivalis is mentioned.

The “ALP activity improving composition” of the present invention refers to S. cerevisiae. mutans and / or P. mutans. It includes gingivalis, and any one that improves the ALP activity of cells. For example, S. cerevisiae that improves ALP activity of human dental pulp-derived fibroblasts (hereinafter referred to as hDPF). mutans and / or P. mutans. A composition containing gingivalis is mentioned.
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.

<Preparation of composition for treatment of hard tissue regeneration>
1. S. A composition for treating hard tissue regeneration comprising mutans a. ~ D. S. A composition for treating hard tissue regeneration containing mutans was prepared.
a. S. inoculated in Todd Height broth (Becton Dickinson and Company). mutans MT8148R was cultured at 37 ° C. for 2 days under anaerobic conditions of 10% CO ₂ , 10% H ₂ and 80% N ₂ .
b. The cells were collected by centrifugation (10,000 × g, 20 minutes, 4 ° C.) and washed twice with PBS (pH 7.4).
c. Using an ultrasonic crusher (Bioruptor UCD-200T, Cosmo Bio Co., Ltd.), the microbial cells were repeatedly crushed for 60 minutes by ultrasonic crushing for 30 seconds and 1 minute intervals.
d. The disrupted extract is centrifuged (10,000 × g, 20 minutes, 4 ° C.) It was used as a hard tissue regeneration treatment composition containing mutans (bacteria crushed material).

2. P. Composition for treating hard tissue regeneration containing gingivalis a. ~ D. P. A composition for treating hard tissue regeneration containing gingivalis was prepared.
a. P. gingivalis ATCC 33277 was inoculated into Tryptic Soy broth supplemented with hemin (5 mg / ml), menadione (0.5 mg / ml) and 10% defibrinated horse blood, 10% CO ₂ , 10% The cells were cultured at 37 ° C. for 2 days under anaerobic conditions of H ₂ and 80% N ₂ .
b. The cells were collected by centrifugation (10,000 × g, 20 minutes, 4 ° C.) and washed twice with PBS (pH 7.4).
c. Using an ultrasonic disrupter (Brason, Danbury, CT, USA), the cells were repeatedly disrupted for 5 minutes at an output of 100 W, an ultrasonic disruption of 30 seconds and an interval of 1 minute.
d. The crushed extract was centrifuged (10,000 × g, 20 minutes, 4 ° C.), and the supernatant was treated with P. a. It was used as a hard tissue regeneration composition containing gingivalis (broken cell).

[Test Example 1]
<Examination of hard tissue regeneration composition>
1. Sample 1) Composition for Regenerating Hard Tissue Regeneration The concentration of protein contained in the crushed cells in each composition for regenerating hard tissue prepared in Example 1 was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). ), And the final concentration of the protein contained in the disrupted cells was 100 μg / ml, 10 μg / ml, 1 μg / ml, 0.1 μg / ml or 0.01 μg / ml. .
2) Lipopolysaccharide (LPS)
LPS produced by E. coli O55 was used as a comparative sample. The LPS concentration was adjusted to 10 μg / ml, 1 μg / ml, 0.1 μg / ml or 0.01 μg / ml and used.

3) hDPF or lower a. ~ E. HDPF was prepared by the following steps.
a. From 3 patients undergoing orthodontics, the third molar of the lower jaw containing hDPF was extracted during the orthodontic treatment and hDPF was collected.
b. The dental pulp tissue was separated from the teeth and subdivided into small pieces of 1 mm ³ or less. Then, an enzyme treatment was performed at 37 ° C. for 20 minutes with an HBS solution (Hank's Balanced Salt Solution) containing 60 U / ml Dispase II (Joint Shusei Co., Ltd.) and 2 mg / ml collagenase (Wako Pure Chemical Industries, Ltd.). .
c. Place a small amount of tissue in a 10 cm culture dish and add 10% FCS serum (JRH Bioscience, Lenexa, KS, USA), glutamine (Gibco, Invitrogen), 100 U / ml penicillin-streptomycin (Gibco, Invitrogen) and 100 μM ascorbic acid Was cultured in a growth medium containing an α-MEM medium (Wako Pure Chemical Industries, Ltd.).
d. Cells sprinkled from the tissue were cultured, the medium was changed every 3 days, and the morphology of the subcultured hDPF was observed with a phase contrast microscope.
e. The confluent cells were detached with 0.05% trypsin and 0.02% EDTA, subcultured, and the second hDPF was used for the test.

2. Test and Results 1) Cell proliferation Cell proliferation was measured using a cell count kit-8 (WST-8; Dojindo, Kumamoto, Japan). That is, the second passage of hDPF was seeded on a culture dish so as to be 1 × 10 ³ cells / well (12 wells), and cultured in a complete medium supplemented with the composition for hard tissue regeneration treatment of the present invention or LPS, respectively. It was. In addition, the culture was performed using a composition for treating hard tissue regeneration or hDPF without addition of LPS as a control. The number of cells on the 14th and 28th days of culture was measured by measuring the number of cells using WST-8 kit (Kishida Chemical Co., Ltd.) at an absorbance of 450 nm (SmartSpec ™ 3000, BIO-RAD).
As a result, S.E. Mutans or P.M. In the composition for treating hard tissue regeneration containing gingivalis, cell proliferation was less than that in the control. In particular, P.I. In the composition for hard tissue regeneration treatment containing gingivalis, the proliferation of cells was significantly low when the concentration of protein contained in the crushed cells was as high as 100 μg / ml.
When LPS was used, the cells proliferated in the same manner as in the control. Mutans or P.M. It was suggested that the growth of hDPF was inhibited in the hard tissue regeneration composition containing gingivalis because of the involvement of LPS in the cell disruption solution.

2) ALP activity The second generation of hDPF was seeded in a culture dish so as to be 3 × 10 ³ cells / well (12 wells), and the growth medium supplemented with the composition for treating hard tissue regeneration or LPS of the present invention was added. Culture was performed. In addition, the culture was performed using a composition for treating hard tissue regeneration or hDPF without addition of LPS as a control. The ALP activity on the 14th and 28th days of culture was measured using the SIGMA-FADT r-nitrophenyl phosphate table set (Sigma, St. Louis, Mo, USA) with an enzyme reaction at an absorbance of 405 nm (SmartSpec ™ 3000, BIO-RAD). Quantified by examining.

As a result, as shown in FIG. In the composition for regenerating and treating hard tissue containing mutans, the concentration of the protein contained in the crushed cell is 10 μg / ml, but the concentration of the protein contained in the control or crushed cell is 100 μg / ml. ALP activity was significantly higher than some. A protein having a concentration of 1 μg / ml, 0.1 μg / ml, or 0.01 μg / ml in the disrupted bacterial cells has a higher ALP activity than these, and therefore, a low concentration of S. cerevisiae. It has been suggested that the composition for treating hard tissue regeneration containing mutans promotes differentiation into odontoblasts or osteoblasts by acting on hDPF and acts on dentin regeneration.
Further, as shown in FIG. When the composition for treating hard tissue regeneration containing gingivalis is used, the concentration of the protein contained in the disrupted bacterial cell is 0.01 μg / ml, while the concentration of the protein contained in the control or disrupted bacterial cell is The ALP activity was significantly higher than that at a high concentration of 100 μg / ml. A protein having a concentration of 10 μg / ml, 1 μg / ml, or 0.1 μg / ml in the disrupted bacterial cells also has a higher ALP activity than these, and therefore, a low concentration of P. coli. It has been suggested that a composition for treating hard tissue regeneration containing gingivalis also promotes differentiation into dentin blasts or osteoblasts by acting on hDPF, and acts on dentin regeneration.

3) Confirmation of expression of hard tissue-forming cell marker gene In the above 2.2), S. cerevisiae was prepared so that the final concentration of the protein contained in the crushed bacterial cells in which an increase in ALP activity was observed was 1 μg / ml. A composition for treating hard tissue regeneration containing mutans and P. cerevisiae prepared so that the final concentration of the protein contained in the crushed bacterial cells is 0.01 μg / ml. The second generation of hDPF was cultured for 10 days in a growth medium to which a composition for treating hard tissue regeneration containing gingivalis was added, and the expression of bone sialprotein (BSP) mRNA, a hard tissue-forming cell marker, was examined. The control was hDPF to which no composition for treating hard tissue regeneration was added.
RNA was extracted from each cell after culturing using Trizol solution (invitrogen), and converted into cDNA by SuperScript III reverse transcriptase (Invitrogen). Thereafter, amplification was performed with Ex Taq DNA polymerase (Takara), and 20 mM primers (SEQ ID NO: 1 and 2 in the sequence listing), and the expression level of BSP mRNA was compared and analyzed.

As a result, BSP expression was not observed in the cells to which the cell disruption solution was not added, but S. cerevisiae prepared so that the final concentration of the protein contained in the cell disruption product was 1 μg / ml. P. mutans-containing composition for hard tissue regeneration or P. cerevisiae prepared so that the final concentration of the protein contained in the disrupted microbial cells is 0.01 μg / ml. It was shown that the expression of BSP increases as shown in FIG. 3 when the composition for treating hard tissue regeneration containing gingivalis is used.
Therefore, from this result, the S.I. A composition for treating hard tissue regeneration comprising mutans and P. mutans It has been suggested that the composition for treating hard tissue regeneration containing gingivalis promotes differentiation into odontoblasts or osteoblasts by acting on hDPF and acts on dentin regeneration.

  Since the composition for regenerating and treating hard tissue of the present invention works for regeneration of dentin and / or periodontal tissue, it can be used as an active ingredient of a pulp capping agent or a therapeutic agent for regenerating bone tissue.

Claims (8)

S. mutans and / or P. mutans. A composition for treating hard tissue regeneration comprising gingivalis. S. mutans and / or P. mutans. The composition for hard tissue regeneration treatment according to claim 1, wherein gingivalis is a crushed cell. S. mutans and / or P. mutans. The composition for treating hard tissue regeneration according to claim 1 or 2, wherein the concentration of the protein contained in the disrupted gingivalis cells is less than 100 µg / ml. The composition for a hard tissue regeneration treatment according to any one of claims 1 to 3, which is used for regeneration of dentin and / or periodontal tissue. A pulp capping agent comprising the composition for treating hard tissue regeneration according to any one of claims 1 to 4 as an active ingredient. A therapeutic agent for regenerating bone tissue comprising the composition for regenerating hard tissue according to any one of claims 1 to 4 as an active ingredient. S. mutans and / or P. mutans. A composition for promoting cell differentiation, comprising gingivalis. S. mutans and / or P. mutans. A composition for improving alkaline phosphatase (ALP) activity of cells containing gingivalis.