JP2672388B2 - Drugs and materials for promoting periodontal tissue regeneration - Google Patents

Description

TECHNICAL FIELD The present invention regenerates periodontitis-disrupted periodontal ligament,
The present invention relates to a drug or medical material used for promoting the adhesion between normal tooth root and connective tissue.

Conventional Techniques and Problems Conventionally, as a method for treating periodontitis, a method of mechanically removing plaque in a periodontal pocket by scaling or the like has been mainly adopted, and a periodontal surgical procedure is used in a serious case. In addition, chemotherapy with antibiotics has been tried recently. However, although these therapies are effective measures to prevent the progression of periodontitis, they do not positively repair and regenerate the destroyed periodontal tissue, and the improvement of clinical symptoms is only a matter of living body. It depends on self-healing power.

Periodontal tissue has a structure not found in other tissues, in which hard tissue (root) and soft tissue (gingiva) are attached by a strong fibrous bond through the periodontal ligament. However, since epithelial cells on the gingival surface cover the wound surface in the periodontal pocket before the periodontal ligament is regenerated (downgrowth of epithelium), only the epithelial tissue and the root are detached. As a result, the periodontal pocket is easily reshaped,
Consequently, recurrence of periodontitis and gingival recession frequently occur.

On the other hand, as a method for achieving normal fibrous binding, (1) root surface treatment with citric acid (2) local application of cell-adhesive glycoprotein fibronectin (3)
A guided tissue regeneration method (GTR method) that suppresses downgrowth of epithelium by a biocompatible blocking membrane has been proposed. However, the method (1) is harmful to cells,
The method (2) has problems such as stability and antigenicity of a high molecular weight fibronectin, and the method (3) physically controls biological functions, so that there is a large difference in success rate among operators.

In view of such circumstances, the present inventors have conducted extensive studies on a periodontal tissue regenerating agent which is excellent in safety, stability and effectiveness and can be easily applied to the induced tissue regeneration method. As a result, they have found that the polyelectrolyte complex has a periodontal tissue regeneration promoting action.

A polyelectrolyte complex is a polymer electrolyte (cationic polymer substance and anionic polymer substance) having opposite signs, which is formed instantly by mixing them as component polymer substances. The interface has a micro-heterogeneous structure in which hydrophobic domains are dispersed in hydrophilic domains mainly having ionic bonds, similar to the surface structure of biological tissue. It has been conventionally known that the use of these polyelectrolyte complexes as a culture medium for animal cells enhances functions such as adhesion of cultured hepatocytes and pancreatic cells, but treatment of periodontitis There is no example of applying these in.

Means for Solving the Problems The present invention is a periodontal tissue regeneration-promoting agent comprising a polyelectrolyte complex formed from an anionic polymer substance and a cationic polymer substance as an active ingredient. And a material for promoting periodontal tissue regeneration, which is obtained by coating a pharmaceutically acceptable base material with the polyelectrolyte complex.

The component polymer substance of the polyelectrolyte complex used in the present invention may be a synthetic substance, a natural substance or a biological substance. As the cationic polymer substance, poly [(dimethyl Main chain charge type strong basic synthetic cationic polymer or poly (vinylbenzyltrimethylammonium chloride) (PVBMA)
Side chain charged type strong basic synthetic cationic polymer substances,
Further, examples of biologically derived cationic polymeric substances include deacetylated N-acetyl-D-glucosamine polymers such as chitosan and its derivatives. In addition, as the anionic polymer substance, synthetic anionic polymer substances having different hydrophobic side chains prepared by radical copolymerization of acrylic acid ester and acrylic acid and hydrophobic chains such as polyacrylic acid are included. Not synthetic anionic polymer,
Furthermore, hyaluronic acid, chondroitin sulfate, alginic acid, cellulose and its derivatives, N-acetyl-
Examples include bio-derived anionic polymeric substances such as carboxymethylated derivatives of chitin, which is a D-glucosamine polymer. In the present invention, it is preferable to use an anionic polymer substance such as a polysaccharide, particularly a bio-derived N-acetyl-D-glucosamine polymer.

A polymer electrolyte complex solution is obtained by preparing an aqueous solution of these component polymer substances into a solution having an ionic group concentration of usually 0.00001 to 0.01 Umol / and mixing them. Further, the charge balance of the obtained polymer electrolyte complex can be changed by appropriately changing this mixing ratio. The obtained polymer electrolyte complex is soluble only in a special ternary solvent, is insoluble in water, organic solvent, acid and alkali, and has high heat resistance and chemical resistance.

Although the safety of these polyelectrolyte complexes varies depending on the type of component polymer substance used and the charge balance,
It is confirmed by trypan blue staining that no toxicity to cells is observed in the concentration range of 10 ^-8 to 10 ^-6 M, which is the concentration range where efficacy is usually observed. Further, since eluates in ordinary solvents cannot be seen, it is not necessary to consider the toxicity of each component polymer substance individually.

Thus, the periodontal tissue regeneration-promoting agent of the present invention is prepared by combining an effective and non-toxic amount of the polyelectrolyte complex with a pharmaceutically acceptable carrier according to a conventional formulation technique. A film formed by a casting treatment of a polyelectrolyte complex, or a medical material (for example, Teflon or silicon), which is coated on a pharmaceutically acceptable substrate such as Teflon or silicon.
Membrane for the guided tissue regeneration method).

Thus, the periodontal tissue regeneration-promoting agent of the present invention can be used by directly applying it to the periodontal pocket after periodontal surgery or root surface lubrication. The applied amount can be appropriately increased or decreased depending on the symptom to be treated and the site, but when it is used as an external preparation, the polyelectrolyte complex is usually used at a concentration of 10 ^-6 to 10 ^-4 M, and the amount is about 0.1 to 0.3 ml once. Inject into the affected area 1 to 3 times a day. Further, the medical material is formed of a Teflon membrane or a silicone membrane whose surface is coated with the polymer electrolyte complex at a concentration of 10 ^-6 to 10 ^-4 M, or the polymer electrolyte complex at a concentration of 10 ^-1 M. A desired periodontal tissue regeneration effect is exhibited by inserting the formed film into the affected area.

EXAMPLES Next, the present invention will be described in more detail with reference to examples and experiments.

Example 1 Component amount Carboxymethylcellulose-chitosan complex (10 ^-2 M) 1 g Sodium lauryl sulfate 0.2 g Glycerin 40 g Purified water 100 g Prepared by mixing these components, a gel agent is obtained.

Example 2 A polycarbonate membrane filter was immersed in an aqueous solution of 2X-polyacrylic acid complex (10 ⁻⁶ M), left standing overnight, taken out, dried in a dryer at 65 ° C. for 3 hours, and then washed with distilled water. Then, it is dried again and sterilized with ultraviolet light to obtain a sterilized membrane filter for regeneration of induced tissue, the surface of which is coated with a 2X-polyacrylic acid complex.

Example 3 A solution of a carboxymethyl chitin-chitosan complex (10 ^-1 M) was formed into a film on silicone or Teflon by a conventional casting treatment, and dried completely (humidity 60 to 70).
%), Washed with distilled water, and sterilized with ultraviolet light to obtain a carboxymethyl chitin-chitosan complex sterilized film. The membrane can be stored in isotonic solutions such as saline or phosphate buffered saline.

Experiment The periodontal tissue regeneration promoting action of a polyelectrolyte complex consisting of a cationic polymer and an anionic polymer was tested. The results are shown below.

(1) Effects on periodontal ligament fibroblast motility Primary periodontal ligament fibroblasts were cultured from the periodontal ligament remaining in human extracted teeth, and their motility was determined by various polyelectrolyte complexes or various component polymeric substances. Surface coated pore size 8
It was measured by a 48-well microchamber method using a micron membrane filter.

5.0 × 10 ⁶ cells / ml cell suspension in the upper chamber,
Only the culture solution was placed in the lower chamber and incubated at 37 ° C. for 4 hours. The filter was then fixed and stained with Diff-Quick, and the number of cells that had migrated to the bottom through the holes of the filter was counted under a microscope. As a control, the same test was performed using a filter whose surface was not coated. Table 1 shows the relative proportions when the polymer electrolyte complex-coated filter was used, where the control count value was 100%.

As shown in Table 1, the motility of periodontal ligament fibroblasts is
On the polymer electrolyte complex-coated filter, it was remarkably enhanced as compared with the case where each component polymer substance was alone. From these results, it is clear that these polyelectrolyte complexes have the action of more selectively migrating periodontal ligament fibroblasts, which are the center of periodontal tissue regeneration, to the lesion site.

(2) Action on Differentiation Ability of Periodontal Ligament Fibroblasts The action of various polyelectrolyte complexes on the differentiation ability of periodontal ligament fibroblasts was measured using alkaline phosphatase activity as an index.

Place the membrane filter whose surface is coated with various polyelectrolyte complexes in a tissue culture dish with a diameter of 35 mm,
3.0x10 ⁴ periodontal ligament fibroblasts were seeded on it, incubated at 37 ° C for 7 days, 0.2% nonidet P-40 treatment was applied to destroy the cells on the membrane filter, and ultrasonic disruption was performed for 10 minutes. The supernatant was obtained by centrifugation. The alkaline phosphatase activity in 0.2 ml of this supernatant was
The amount of p-nitrophenol produced by the enzymatic reaction (30 minutes) using disodium p-nitrophenylphosphate as a substrate was quantified by measuring the absorbance (A ₄₁₀ ) at ₄₁₀ nm. As a negative control, a membrane filter whose surface was not coated, and as a positive control, dexamethasone was further added to the medium so that the final concentration was 10 μg / ml, and the same test was conducted. Further, the same test was performed on the action of each polymer substance alone of the polymer electrolyte complex.

The enzyme unit (1 unit) is p at 1 minute at 37 ℃.
The specific activity was calculated by the following formula, with the amount of 1 nmol of nitrophenol produced.

Specific activity (unit / mg protein) = A ₄₁₀ x 100 / (0.873 x 0.2 ml x 30 minutes x protein amount) The results are shown in Table 2.

As shown in Table 2, the polyelectrolyte complex enhances the alkaline phosphatase activity of periodontal ligament fibroblast cells and enhances the cell differentiation ability. Clearly from this result,
The polyelectrolyte complex has the activity of further differentiating locally migrated periodontal ligament fibroblasts to form normal periodontal ligament fibers.

(3) Effect on periodontal tissue regeneration process after canine gingival ablation curettage Guided tissue regeneration method (GTR method) after canine gingival ablation curettage surgery
The effects of various polyelectrolyte complexes on the treatment process by PT were investigated by histopathological quantification method. The upper and lower premolars with healthy periodontal tissues established by brushing etc. were subjected to gingival dissection curettage surgery according to a standard method. At this time, a reference point called a notch was added to the root surface before and after the control of the alveolar bone to serve as a reference point for later histopathological quantification. The sample was a membrane filter whose surface was coated with a polymer electrolyte complex similar to that shown in Example 2, and the filter was applied to the test site of the left and right upper and lower jaws according to the GTR method. As a control, a filter having an uncoated surface was applied to the upper and lower right jaws. After surgery, the flap was repositioned and protected with sutures and packs for 1 week. The evaluation was conducted 3 months after the surgery, the site to be examined was sampled, and a tissue sample was prepared by a conventional method.
The distance between each site was measured using an eyepiece micrometer under a microscope, and calculated by the following formula.

Table 3 shows the results.

As shown in Table 3, in regeneration of periodontal tissue by the GTR method, the filter coated with the polyelectrolyte complex significantly promoted the formation of new cementum as compared with the control filter. The suppression of downgrowth was similar.

As is clear from the above results, the polyelectrolyte complex has an excellent periodontal tissue regeneration promoting action.

EFFECTS OF THE INVENTION According to the present invention, periodontal tissue regeneration-promoting agents and materials which are useful for the treatment of periodontitis and which are excellent in safety, stability and effectiveness are obtained.

Continuation of the front page (72) Inventor Miwako Ureshino 2-10-1 Kamitomuro, Takatsuki City, Osaka Prefecture

Claims (2)

(57) [Claims] 1. A periodontal tissue regeneration-promoting agent comprising a polymer electrolyte complex formed from a cationic polymer substance and an anionic polymer substance as an active ingredient. 2. A material for promoting periodontal tissue regeneration, which is obtained by coating a pharmaceutically acceptable base material with a polyelectrolyte complex formed of a cationic polymer substance and an anionic polymer substance.