JP5215546B2 - Periodontal tissue regeneration composition - Google Patents

Description

  The present invention relates to a periodontal tissue regeneration material. More specifically, the present invention relates to a composition for periodontal tissue regeneration that is effective for reconstituting periodontal tissue cells in the initial stage of regeneration by causing periodontal ligament cells to migrate to the affected area.

  Periodontal disease is one of the two major dental diseases along with dental caries. Endotoxin produced by pathogenic bacteria such as Porphyromonas gingivalis causes inflammation of periodontal tissues such as gums, periodontal ligament, cementum and alveolar bone. It is a disease that causes a reaction and causes tissue absorption. This causes tooth swaying, and if it becomes severe, the tooth falls off. Examples of treatments that have been performed clinically for the purpose of recovering lost periodontal tissue for patients with moderate or higher periodontal disease include flap surgery and GTR (tissue-guided regeneration).

  In the flap technique, the affected gingiva is once removed, the infected root is cut with a surgical instrument, the infected soft tissue is excised, and the gingiva is sutured again. Healthy periodontium has a unique structure in which hard tissues (roots, especially cementum and alveolar bone) and soft tissues adhere to each other through a strong fibrous bond. The mode of connection between tissues is that epithelial cells grow faster than other cells, resulting in adhesion with low adhesive strength, periodontal ligaments cannot be regenerated, and gingival retraction occurs frequently. Become. For this reason, a periodontal pocket is easy to form again, and it becomes a hotbed of a pathogenic microbe, Therefore The possibility of the re-morbidity of periodontal disease becomes high.

  On the other hand, in the GTR method, a bioabsorbable or non-absorbable membrane is placed between the root and soft tissue after removal of the infected tissue in order to avoid epithelial adhesion between the tooth and soft tissue obtained by the flap technique. After that, the surgical method to be sutured is taken. As a result, proliferation of the epithelial cells to the root can be avoided by the membrane, and the root and soft tissue are firmly bonded via the mesenchymal cells.

  However, GTR treatment also has the advantage that the adhesion between the teeth and soft tissue is good and the recurrence of the periodontal pocket is less likely to occur, but the time required for periodontal tissue regeneration is long, so the membrane in between Infection is a problem, the difference in the success rate by the surgeon, the difference in the degree of improvement by the case is a problem, and further, the bone root causes bone adhesion, The regeneration of cementum is very slow, and there are many cases in which healthy periodontal tissue cannot be obtained as a result, such as normal planting of teeth through cementum-periodontal ligament-alveolar bone is not performed. It is mentioned as.

  As a means for solving these problems, Patent Document 1 proposes regeneration of periodontal tissue with an active enamel substance, but it is said that it has cleared the safety problem caused by the animal from which it originated. hard.

Furthermore, in Non-Patent Document 1, it is proposed to regenerate periodontal tissue using platelet-derived growth factor, but this growth factor has a high production cost or exhibits its effect at a relatively high concentration. Therefore, it has various problems such as cost.
Special Table 2002-504520 L. A. Boyan et al. Journal of Dental research 73 (10): 1593-1600

  An object of the present invention is to provide a material that can efficiently regenerate periodontal tissue affected by periodontal disease into a healthy state.

  In periodontal tissues, periodontal ligament cells are classified into various types of cell populations. These include cells that differentiate into cementum, cells that form periodontal ligament fibers, and cells that differentiate into alveolar bone. During regeneration of periodontal tissue, lost periodontal ligament cells are transferred to the affected area. Is the first key to regeneration leading to subsequent proliferation and differentiation.

In the composition for periodontal tissue regeneration of the present invention, the culture medium is divided into upper and lower parts by a porous membrane having pores through which cells can pass, and a cell suspension of human periodontal ligament cells is placed on the upper part, and the lower part is placed on the lower part. When a medium containing a chemotactic substance at a concentration of 1 ng / ml is placed, the chemotaxis has a chemotaxis of 1.5 or more in the fluorescence intensity ratio of cell staining after 22 hours compared to the case where no chemical is added. It is characterized by containing enolase , which is a chemical substance, at least a factor that causes migration of periodontal ligament cells.

  In the present invention, enolase, which is a factor that migrates periodontal ligament cells, is a glycolytic enzyme present in the cytoplasm, and is expressed specifically in neurons, and has been reported to be associated with Alzheimer's disease, for example. The relationship with tissue cells has never been known.

  As a result of intensive studies by the inventors, among the gingival cells, alveolar bone cells, and periodontal ligament cells constituting periodontal tissue, enolase specifically and specifically selects periodontal ligament cells with high selectivity at a surprisingly low concentration. It has been found that there is a possibility of promoting local migration, and the present invention has been completed.

  By using the periodontal tissue regeneration composition of the present invention, it becomes possible to regenerate periodontal tissue affected by periodontal disease into a healthy tissue.

The present invention is described in detail below. Unless otherwise specified, “part” or “%” indicates a weight basis.
In order to achieve the above object, the medium is divided vertically with a porous membrane having pores through which cells can pass, human periodontal ligament cells are placed in the upper part, and chemotaxis at a concentration of 1 ng / ml in the lower part. Fluorescence intensity ratio of cell staining after 22 hours [(fluorescence intensity when chemotaxis substance is blended) / (when no chemotaxis substance is blended] It is important to use a chemotactic substance having a chemotaxis of (fluorescence intensity), hereinafter referred to as a chemotaxis index in this specification) of 1.5 or more, preferably 1.6 or more, more preferably 1.7 or more. It is.

  In addition to periodontal ligament cells, the chemotaxis index for other cells (gingival fibroblasts, alveolar bone-derived osteoblasts, gingival epithelial cells) that constitute periodontal tissue is chemotaxis to periodontal ligament cells. Ratio to sex index ((chemotaxis index for cells other than periodontal ligament cells constituting periodontal tissue) / (chemotaxis index for periodontal ligament cells)) is usually less than 0.75, preferably 0.7 Below, more preferably 0.67 or less.

The chemotaxis measurement conditions are those that can be measured by a normal measurement method in the technical field, and are not particularly limited, but a suitable measurement method is described below. That is, a Dulbecco's modified Eagle medium (hereinafter referred to as DMEM in the present specification) containing a test substance at the bottom, and 5 × 10 ⁴ cells at the top through a porous membrane having a pore size of 8 to 12 μm and no cytotoxicity. A suspension of DMEM containing human periodontal ligament cells is placed and cultured at 37 ° C. for 22 hours. Subsequently, after the cells non-specifically adhered to the porous membrane are washed, the cells are stained with a fluorescent dye, and the fluorescence intensity is measured. The chemotaxis can be measured by dividing the obtained value by the fluorescence intensity when no test substance is contained. The test conditions can be easily reproduced by using a fluoroblock insert system (trade name, manufactured by Becton Dickinson).

  A non-cytotoxic membrane having a pore size of 8 to 12 μm is used to prevent non-specific migration of test cells with respect to substances other than the chemotactic activity test substance, and can be replaced if it has an equivalent function. is there. DMEM is used for culturing the cells and can be substituted if it has an equivalent function.

Cell staining for counting the number of cells includes, for example, direct observation with a phase contrast microscope, Papanicolaou staining, hematoxylin or hematoxylin-eosin double staining, and fluorescence staining with calsein-AM can be preferably selected. . At this time, it is preferable that the measurement light beam is not greatly separated from the excitation and fluorescence center wavelength of the fluorescent staining reagent. In the case of Calsein-AM, it is desirable to select 460 to 510 nm as the excitation wavelength and 490 to 540 nm as the fluorescence wavelength, and the excitation wavelength is shorter than the fluorescence wavelength.

  Heretofore, no chemotactic substance having such a high activity has been found to be practical, but it has been found that, among glycolytic enzymes, enolase is particularly highly active in periodontal ligament cells.

  In the present invention, the type of enolase used as a factor for migrating periodontal ligament cells is not particularly limited, but those containing a γ subunit are preferred. If it contains a γ subunit, it can act as a monomer, form a homodimer of the γ subunit (γγ type), or a heterodimer (αγ type, βγ type) with subunits such as α and β In the present invention, a homodimer of a γ subunit or a heterodimer of an α subunit and a γ subunit is particularly preferable.

The enolase in the present invention preferably has a human amino acid sequence. In addition to those extracted from humans, the amino acid sequence or cDNA sequence is known (for example, GenBank / NM_001975). Release isolated cDNA clones in vitro
It can also be prepared and used after being expressed in a transcription / translation system or a suitable host vector system. Necessary genetic engineering or molecular biological techniques are described in, for example, Sambrook and Russell, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press
, 2001 etc. Furthermore, peptides can be synthesized and used using a known amino acid solid phase synthesis method or the like.

  The composition for periodontal tissue regeneration of the present invention is prepared by combining an effective and non-toxic amount of the enolase with a pharmaceutically acceptable carrier such as a solvent, a suspending agent, a stabilizer and the like according to a conventional formulation technique. Can be In addition, collagen, gelatin, other natural polymer compounds, and further synthetic polymer compounds can be blended as the enolase scaffolding material to prepare a preparation. Normally, when the enolase is applied to the root surface at a concentration of 10 fg to 10 μg per 1 ml of body fluid at the periodontal site to be treated, a desired periodontal tissue regeneration effect is exhibited.

  The dose and duration for applying enolase to periodontal ligament cells and regenerating periodontal tissue is determined by the disease state of the regenerated periodontal tissue. The application period is usually several hours to several days, and can be longer.

  The exact effective amount for a human subject is the severity of the disease state, the general health of the subject, the subject's age, weight and gender, diet, application time and frequency, concomitant medications, sensitivity to response, and tolerance to treatment It depends on sex and reaction.

  This dose can be determined by routine experimentation and, if used clinically, is within the judgment of the physician or dentist. In general, the effective amount per body weight is 0.001 mg / kg to 50 mg / kg. However, when enolase is introduced in a locally concentrated form, for example, when it is embedded in a periodontal tissue while being supported on a membrane, the effective amount may be higher than this.

In vitro, enolase is exposed to periodontal ligament cells in cell cultures in the range of 10 fg / ml to 10 μg / ml, preferably 100 fg / ml to 5 μg / ml, more preferably 1 pg / ml to 1 μg / ml. May be. Therefore, the chemotactic substance is preferably 1 × 10 ⁻⁶ to 1%, more preferably 1 × 10 ^{−5 to} 0.5%, and even more preferably 1 × 10 ^{6 in the} composition for periodontal tissue regeneration. ^{-4 to} 0.1%. When the composition for periodontal tissue regeneration of the present invention is applied and prepared for the purpose of sustained release of the chemotactic substance over a long period of time, these upper limit values may be higher, but the effective sustained release amount is Preferably no more than 10 μg per ml of body fluid.

  As the periodontal ligament cells used for the evaluation of the composition for periodontal tissue regeneration of the present invention, cells isolated by a conventionally known method can be passaged and used. Specific examples include the methods described below. Tooth removed for convenience during orthodontic treatment is washed with DMEM containing antibiotics such as amphotericin B and penicillin and fetal bovine serum, and periodontal tissue attached to the teeth is removed with a dental scaler, etc. And then cultivated at 37 ° C. for 1 day using the same medium. Subsequently, the cells grown around the tissue piece are detached from the culture dish using an enzyme such as trypsin, and this is used as a primary cell. In general, the passage number can be up to about nine.

  As a method for evaluating the composition for periodontal tissue regeneration of the present invention, a conventionally known method can be used. Specifically, a culture medium containing a test substance is prepared in a culture dish, and a basket with a membrane attached to the bottom is placed on the culture dish for testing, or an in vitro test method represented by a so-called Boyden chamber And in vitro test methods using bromodeoxyuridine and the like. Among these, an in vitro test method can be preferably selected for reasons such as simplicity of the test, and in the in vitro test method, the time for cell migration is generally in the range of 1 to 24 hours.

Next, the carrier that can be used in the composition for periodontal tissue regeneration of the present invention will be described.
As the solvent, conventionally known solvents can be used as long as they are pharmaceutically acceptable, and specific examples include water, physiological saline, phosphate buffered saline (PBS), glycerol, and the like. Can do.

Moreover, conventionally well-known substances can be used for the collagen used here, These can also be used individually or in combination of 2 or more types. Specifically, Type 1, Type 2, Type 3, Type 4, Type 5, Type 6, Type 7, Type 8, Type 9, Type 10, Type 11, Type 12, Type 13, Type 14, Type 15, Type 16, Type 17, Type 18, Type 19, Type 20, Type 21, Type 23, Type 24, and the like. Any organism can be used as a source organism, such as mammals such as humans, cows and pigs, fish such as salmon, and recombinants using fungi such as Escherichia coli. Among these, it is preferable to use salmon-derived collagen in consideration of safety to the human body. In addition, these collagens are preferably atelolated in order to reduce the harmfulness to tissues. In order to further increase the strength of the collagen, it is preferable to perform a crosslinking treatment. As a crosslinking method, a conventionally known method can be selected. Specifically, physical crosslinking such as ultraviolet crosslinking, water-soluble carbodiimide crosslinking using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, glutar, and the like. Examples thereof include chemical crosslinking such as aldehyde coupling and enzymatic crosslinking such as transglutaminase crosslinking.

  Conventionally known gelatin can be used. Specifically, it can be used by producing the above collagen as a starting material using normal gelatin industrially obtained from pig skin or the like by an alkali method or an acidic method, or a conventionally known method.

  Furthermore, it is preferable to perform a crosslinking treatment on gelatin in order to increase the strength, like the above-described collagen. As a crosslinking method, a conventionally known method can be selected, but a heat crosslinking method using a crosslinking agent can be preferably selected. As the crosslinking agent, for example, a water-soluble epoxy compound, a water-soluble aldehyde, or a water-soluble carbodiimide can be used. Examples of water-soluble epoxy compounds include glycerol polyglycidyl ether, sorbitol polyglycidyl ether, polyglycerol polyglycidyl ether, ethylene glycol polyglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, propylene glycol diglycidyl ether. be able to. Examples of water-soluble aldehydes include glutaraldehyde, formaldehyde, and glyoxal. As an example of water-soluble carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride can be mentioned. Among these, water-soluble epoxy compounds are preferable because they are easy to handle and have low toxicity, and glycerol polyglycidyl ether is particularly preferable.

  The amount of the crosslinking agent used varies depending on the type of the crosslinking agent, the type of gelatin, etc., but is usually 1 to 20% of the total amount of gelatin, and the crosslinking temperature and time are generally 80 to 150 ° C. and 1 to 24, respectively. It is a range of time.

  A conventionally well-known substance can be used as a natural high molecular compound, and it can also be used only by 1 type or in combination of 2 or more types. Specific examples of compound names include cellulose, amylose, agar, and alginic acid.

As the synthetic polymer compound, a conventionally known substance can be used, and it can be used alone or in combination of two or more. Specific compound names include
Bioabsorbable synthetic polymers such as polylactic acid, polyglycolic acid, lactic acid glycolic acid copolymer, polyhydroxyalkanoate, polyamino acids synthesized from essential amino acids only;
Polyethylene glycol, polypropylene glycol, polyvinyl pyrrolidone, poly (N-isopropyl (meth) acrylamide) (hereinafter referred to as “(meth) acryl ...” as a prefix indicating acrylic, methacryl, acrylate, methacrylate, etc.) Water-soluble synthetic polymers such as
Water-insoluble and non-bioabsorbable synthetic polymers such as poly (alkyl (meth) acrylate), polyethylene, polypropylene, poly (tetrafluoroethylene), polyvinylidene difluoride, etc .;
Examples thereof include compounds obtained by using the above-mentioned natural polymer compounds such as carboxymethyl cellulose and propylene glycol alginate as chemical starting materials.

  It is particularly desirable to select and use those compounds having biocompatibility, and these compounds may be molded into a thin film for the purpose of compatibility with tissue or used in the form of a porous film. In the case of having a film-like form, the thickness is preferably 2 mm or less, particularly 0.1 to 1.0 mm, in order to facilitate the operability of the film and the entry of cells into the root surface.

Examples of the composition for periodontal tissue regeneration of the present invention include the following examples.
(1) At the time of use, enolase is mixed with a solvent such as water and applied directly to the necessary site;
(2) Directly dripping with a syringe filled with a solution in which enolase is dissolved in advance, or injecting into the affected area;
(3) Using a carrier, a gel in which enolase is dissolved is prepared in advance and applied directly to the necessary site. (4) Enolase and the carrier are mixed in the presence of a solvent in advance, and formed into a film by lyophilization or the like. Used as a kind of GTR film

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples.
<Preparation of cell suspension>
The cells are cultured in a medium supplemented with 10% fetal bovine serum, cells in logarithmic growth phase are detached with 0.25% trypsin / EDTA (manufactured by Invitrogen), rinsed with DMEM not containing phenol red, 5 × 10 ⁴ cells / A ml cell suspension was prepared.
<Cell chemotaxis test>
500 μl of the cell suspension was placed in the upper chamber of the fluoroblock insert (Becton Dickinson Biosystem), and 500 μl of DMEM containing the test substance was placed in the lower chamber, and left at 37 ° C. for 20-22 hours. Thereafter, the filter is taken out from the upper chamber, washed with physiological saline, stained with Calsein-AM (Molecular Probes), and fluorescent with an excitation wavelength of 485 nm and a fluorescence wavelength of 520 nm using SpectaFluor Plus (manufactured by Tecan). The strength was measured. By this method, the number of cells attracted to the test substance in the lower chamber and chemotactic can be measured from the magnitude of the measured fluorescence intensity, and the chemotaxis activity can be evaluated. Even when no chemotaxis inducer is added to the lower chamber, fluorescence intensity may be measured for non-specific cell migration, so when only DMEM was added to the lower chamber and the test was performed The chemotaxis index was calculated and evaluated by the value divided by the fluorescence intensity of (the fluorescence intensity when the test substance was contained in the lower chamber / the fluorescence intensity when the test substance was not contained in the lower chamber).
[Example 1]
Human periodontal ligament fibroblasts (Cambex) cultured in DMEM supplemented with 10% fetal bovine serum, cells from passage 5 to 8 were used as described above from cells in logarithmic growth phase. A cell suspension was prepared.

Using the DMEM in which 100 pg / ml to 1000 ng / ml of human γγ-enolase (Neuron specific enolase, Human brain, Calbiochem) was dissolved as a test substance, the above-mentioned periodontal ligament cell chemotaxis test was performed. The results are shown in Table 1.
[Example 2]
A cell chemotaxis test of periodontal ligament cells was performed in the same manner as in Example 1 except that human αα-enolase (Non-neuronal enolase, manufactured by Biodesign) was used instead of human γγ-enolase. The results are shown in Table 1.

  By using the periodontal tissue regeneration composition of the present invention, it becomes possible to regenerate periodontal tissue affected by periodontal disease into a healthy tissue.

Claims (3)

  The medium is divided into upper and lower parts using a porous membrane having pores through which cells can pass, a cell suspension of human periodontal ligament cells is placed at the top, and a chemotactic substance with a concentration of 1 ng / ml is blended at the bottom. When the medium is disposed, at least periodontal ligament cells, which are chemotactic substances having a chemotaxis of 1.5 or more in the fluorescence intensity ratio of cell staining after 22 hours, are migrated as compared with the case where no medium is added. A composition for periodontal tissue regeneration comprising enolase as a factor.   2. The periodontal tissue regeneration composition according to claim 1, wherein the factor that causes migration of periodontal ligament cells is enolase containing a γ subunit.   The periodontal tissue regeneration composition according to claim 1, wherein the periodontal tissue regeneration composition further comprises at least one selected from the group consisting of collagen, gelatin, natural polymer compounds, and synthetic polymer compounds. Composition.