JP4575687B2 - Dentinogenic pulp capping agent - Google Patents

Description

  The present invention relates to a dentine-forming pulp capping agent that protects exposed pulp surfaces and promotes dentin formation.

  The pulp is a vascular connective tissue that fills the cavity of the pulp cavity inside the tooth, and is a non-calcified tissue formed from a tooth papilla derived from the mesoderm. In contrast, dentin is a hard calcified tissue around the pulp cavity. Traditionally, the dental pulp has been treated as completely different from the structural features of soft tissue and the dentin as hard tissue. However, in recent years, the pulp and dentin are homologous in terms of developmental and functional homology, and tend to be regarded as “Dentin-Pulp Complex” in the clinic.

  The pulp has various functions such as defense against external stimuli such as infection, perception and transmission of sensory organs, maintenance of dentin homeostasis (dentin formation and nutrient supply), root formation, etc. It is an important organization.

  When a tooth suffers from caries, treatment to protect without removing the pulp (direct pulp capping), removal of only a part of the pulp (crown pulp) and preservation of the root pulp (life pulp cutting) ), Removing the entire pulp and sealing the cavity with metal or resin (pulping). However, after removal of the pulp, the supply of nutrients to the teeth is cut off, dentin weakens, and there is no nerve that conveys pain, so when caries progresses again, subjective symptoms are not obtained and it gets worse There's a problem. Therefore, in recent years, it has been considered preferable to preserve the dental pulp as much as possible in order to maintain dental health. In order to preserve the pulp and maintain its function, indirect or direct pulp capping agents are used, depending on the dentin breakage and the pulp pathological state. Indirect pulp capping is used when the dentin is damaged but the pulp is not exposed, and direct pulp capping cuts part of the pulp when the pulp is exposed or treatment Used in cases. Conventionally, calcium hydroxide preparations and formcresol preparations have been used as direct capping agents. However, calcium hydroxide has no odontoblast-inducing action and cannot promote dentin formation. Up to now, bovine blood extract (Patent Document 1), polysaccharides such as N-acetyl-glucosamine (Patent Document 2), bone formation factor (Patent Document 3), etc. have been reported as having dentin formation promoting action. Has been. However, when bovine blood extract is used, the possibility of prion infection such as mad cow disease or unknown virus infection through the extract to be used cannot be denied, and it should be used for human safety. It is difficult. In addition, when N-acetylglucosamine is used, it is considered safer than the bovine blood extract. However, biological tissues such as insects and crustacean shells are used as raw materials. The influence by cannot be denied. In the method using bone morphogenetic factors such as BMP (bone morphogenetic protein), human-derived recombinant protein is an active ingredient, the preparation method is complicated, the yield of active ingredient is poor, and the product is extremely expensive to manufacture. Probability is high. BMP also has physiological functions other than dentin regeneration, and it cannot be denied that unknown side effects are caused by local large-scale administration.

  On the other hand, polyphosphoric acid is a substance that is originally contained in tissues and cells of many biological species and is always synthesized in vivo (see Non-Patent Document 1). In addition, the safety of polyphosphoric acid for living bodies has been confirmed for a long time, and it is known that it is a biodegradable substance that can be decomposed into nontoxic phosphoric acid in vivo. In addition, polyphosphoric acid can be easily chemically synthesized and can be obtained at a very low cost from a highly pure raw material. Although the physiological function of polyphosphate is unknown, we have stabilized a physiologically active protein such as cell growth factor such as FGF in the polyphosphate by our series of studies on polyphosphate. It was found that there is a function to control. Specifically, a cultured cell growth promoting action, a tissue regeneration promoting action (Patent Document 4, Non-Patent Document 2), a calcification promoting action, and a bone differentiation induction promoting action (Patent Document 5) have been confirmed. As a result of further research, it has been proposed to use a complex with collagen in order to effectively exert the tissue regeneration promoting action of polyphosphate (Patent Document 6). However, until now, the research in the dental field related to the calcification-promoting action and the bone differentiation-inducing action of polyphosphate has mainly been related to the restoration of the dental calcaneus or the jawbone, and the tissue restoration and regeneration of the teeth (dentin) itself There are no reports.

JP 2002-363084 A Japanese Patent Laid-Open No. 06-256132 Japanese Patent Laid-Open No. 06-340555 JP2000-069961 JP 2000-79161 A JP 2004-000543 A H. C. Schroder et al., Inorganic polyphosphate in eukaryotes: Enzymes, metabolism and function, Progress in Molecular and Subcellular Biology, Vol. 23, 45-81, 1999 T. Shiba et al., Modulation of Mitogenic activity of fibroblast growth factors by inorganic polyphosphate, The Journal of Biological Chemistry, Vol. 278, pp.26788-26792, 2003

  The subject of this invention is providing the chemical | medical agent which can accelerate | stimulate formation of dentine while protecting the exposed pulp surface in the dental pulp treatment of a caries treatment.

  As a result of intensive studies to solve the above-described problems, the present inventors have an effect of promoting the formation of dentin when biocompatible and highly safe polyphosphoric acid is applied to the exposed pulp. As a result, the present invention has been completed.

That is, the present invention includes the following inventions.
(1) A dentin-forming pulp capping agent containing polyphosphoric acid as an active ingredient.
(2) Polyphosphoric acid has the following general formula (I):
H _{n + 2} (P _n O _{3n + 1} ) (I)
(Wherein n represents an integer of 3 to 800), and the dentin-forming pulp capping agent according to (1), which is a mixture of one or more of linear phosphoric acids.
(3) The dentine-forming pulp capping agent according to (2), wherein n in the formula is an integer of 40 to 150.
(4) The dentine-forming pulp capping agent according to any one of (1) to (3), wherein the polyphosphoric acid is a polyphosphate.

  ADVANTAGE OF THE INVENTION According to this invention, when it applies to the pulp surface exposed after the pulp treatment in a caries treatment, the dentine formation pulp capping agent which not only protects a dental pulp but can promote formation of a dentine is provided. The dentine-forming pulp capping agent of the present invention has biocompatibility and biodegradability, and is excellent in safety.

  The present invention provides a drug (hereinafter referred to as “dentin-forming pulp capping agent”) that, when applied to an exposed pulp, can not only protect the pulp but also promote the formation of dentin. In the present invention, “dentin formation” includes both regeneration and renewal of dentin. The term “dentin” includes both the second dentin and the third dentin (restored dentin), but mainly refers to the second dentin.

The dentine-forming pulp capping agent of the present invention contains polyphosphoric acid as an active ingredient.
The polyphosphoric acid used in the present invention is typically a linear polyphosphoric acid having a structure in which two or more PO ₄ tetrahedrons share an apex oxygen atom and are linearly linked by dehydration condensation of orthophosphoric acid. Although it is an acid, the side chain polyphosphoric acid by which the organic group was introduce | transduced into the side chain, cyclic polyphosphoric acid, and polyphosphoric acid (ultra polyphosphoric acid) which is a branched phosphoric acid polymer may be sufficient.

The polyphosphoric acid particularly preferably used in the present invention is represented by the general formula (I):
H _{n + 2} (P _n O _{3n + 1} ) (I)
(In the formula, n represents an integer of 3 to 800), and one or a mixture of two or more selected from linear phosphoric acid.

  N in the above general formula is an integer of 3 to 800, preferably 30 to 500, more preferably 40 to 150.

  Polyphosphoric acid having a chain length of 1000 or more has not been confirmed to exist in the form of an aqueous solution, and is considered to be hardly soluble in water, which is not preferable. In addition, since the chain length of polyphosphoric acid is about 800 in vivo, it is considered that polyphosphoric acid having a chain length of 800 or less has high effectiveness for various physiological functions in vivo (KDKumble and A. Kornberg , Inorganic polyphosphate in mammalian cells and tissues, The Journal of Biological Chemistry, Vol.270, pp.5818-5822, 1995).

  Moreover, in this invention, you may use the polyphosphate which has the molecular structure which the hydrogen of the hydroxyl group of said polyphosphoric acid substituted with the metal, and sodium, potassium, calcium, magnesium etc. are mentioned as a metal.

  The polyphosphoric acid or salt thereof used in the present invention may be one kind, but may be a mixture of plural kinds. The plural types of polyphosphoric acids or salts thereof include polyphosphoric acids or salts thereof having different degrees of polymerization, polyphosphoric acids or salts thereof having different molecular structures, and polyphosphates having different metal ions. Moreover, you may include both polyphosphoric acid and its salt.

  Said polyphosphoric acid can be manufactured by the manufacturing method used normally, such as the method of heating phosphoric acid, the method of adding and dissolving phosphorus pentoxide in phosphoric acid.

  Particularly, medium-long chain polyphosphoric acid having a chain length of 20 or more can be produced by the following method developed by the present inventors. First, hexametaphosphate is dissolved in water so as to be 0.1 to 10% by weight, preferably 10% by weight. In this hexametaphosphoric acid aqueous solution, 87-100% ethanol, preferably 96% ethanol, is added in an amount of 1/10 to 1/3 of the total liquid volume after mixing the hexametaphosphoric acid solution and ethanol. Add in an amount to give a volume ratio of 2: 1 to 9: 1. The mixed solution is sufficiently stirred, and the resulting precipitate is separated from the aqueous solution component using a separation method such as, but not limited to, centrifugation or filter filtration. The precipitate thus separated is medium-long chain polyphosphoric acid. The polyphosphoric acid is subsequently washed with 70% ethanol and then dried. The average chain length of polyphosphoric acid obtained by such a separation operation is 60 to 70, and 10 or less short-chain polyphosphoric acid is hardly contained. Therefore, the molecular weight distribution is about 10 to 150 in terms of the number of phosphate residues.

  The content of polyphosphoric acid in the dentine-forming pulp capping agent of the present invention is not particularly limited, but for example 0.001 to 20% by weight, preferably 0.01 to 10% by weight, more preferably 0.1 to 5% by weight, most preferably 0.2. It may be ˜2% by weight.

  Polyphosphoric acid or a salt thereof can be formulated into various preparations in a form suitable for application to the affected area by itself or mixed with pharmacologically and pharmaceutically acceptable additives. Examples of the dosage form suitable for the dentine-forming pulp capping agent of the present invention include, for example, injections, external liquids (injections, coating agents), solid preparations (granules, fine granules, powders, ointments, tablets), It can be suitably prepared by a known method in the form of an ointment or the like.

  Examples of pharmacologically and pharmaceutically acceptable additives include excipients, disintegrating agents or disintegrating aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers, or the like. Solubilizing agents, isotonic agents, pH adjusters, stabilizers, preservatives, preservatives, dispersants, emulsifiers, gelling agents, thickener adhesives, flavoring agents and the like can be used.

  The gelling agent may be, for example, a gelling agent that absorbs tooth exudate. Moreover, you may mix and knead | mix and use as a form which consists of a powder agent and a liquid agent.

  The dentine-forming pulp capping agent of the present invention may further contain other active ingredients such as bactericides, antibiotics and anti-inflammatory agents.

  The dentin capping agent of the present invention can be used by applying or filling a normal caries treatment, for example, opening of the medullary cavity, and filling on the cut surface of the cavity of the cavity after extraction.

  The dentine-forming pulp capping agent of the present invention is used in combination with a direct capping agent or an indirect capping agent, even if applied before or after the application, or mixed with a direct capping agent or an indirect capping agent. May be. In the present specification, the “direct pulp capping agent” is a drug that protects the dental pulp tissue when part of the dental pulp is exposed, and for example, a calcium hydroxide preparation is used. “Indirect pulp capping agent” is a drug used for the purpose of blocking external stimuli and sterilization when the dentin is thin but the pulp is not exposed. For example, zinc oxide Eugenol preparation, zinc oxide cleo Sort preparations are used.

  The dentin-forming pulp capping agent of the present invention facilitates the application of the active ingredient of the drug to the affected area and allows the active ingredient to be retained in the affected area for a period sufficient for dentin formation. It may be supported. Therefore, the dentine-forming pulp capping agent of the present invention is a dental material in which polyphosphoric acid is fixed or impregnated in a structure having a shape such as monofilament, film, fiber aggregate, sponge, and fine particles together with a suitable auxiliary agent. Form may be sufficient.

  The dose of the dentine-forming pulp capping agent of the present invention is not particularly limited and can be appropriately adjusted depending on the patient's symptoms (caries progression), age, dosage form, etc. For example, the active ingredient polyphosphate is added. The dry weight may be 1 ng to 100 μg, preferably 10 ng to 10 μg per time.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1
A 500 mM sodium polyphosphate solution was prepared by dissolving 51 mg of sodium polyphosphate stock (average chain length 40) in 1 ml of sterile water.

(Comparative Example 1)
A 500 mM sodium phosphate buffer solution was prepared by appropriately mixing disodium hydrogen phosphate having the same concentration as 500 mM sodium dihydrogen phosphate so as to have a pH of 7.0.

(Comparative Example 2)
108 mg of β-glycerophosphate and 125 mg of ascorbic acid were dissolved in 1 ml of sterilized water, respectively, to obtain a 2.5M β-glycerophosphate solution and a 1.25% ascorbic acid solution.

(Test Example 1) Calcification induction test of human odontoblasts Using odontoblasts isolated from human (8-year-old boy) deciduous dental pulp, each composition prepared above was used as a test substance, and odontoblasts using these compositions The calcification induction effect was investigated. Odontoblasts were seeded on a cell culture dish having a diameter of 60 mm, and cultured to a confluent state using an α-MEM medium containing 10% bovine serum. Thereafter, 2 μl of the sodium polyphosphate solution prepared in Example 1 and the sodium phosphate buffer solution prepared in Comparative Example 1 were each prepared in Comparative Example 2 in α-MEM medium containing 1% bovine serum. 4 μl of glycerophosphate solution and ascorbic acid solution were added to each 1 ml of the culture solution to induce calcification of odontoblasts. The medium was changed every 4 days. (1) Change in the mRNA level of cell calcification marker genes (osteopontin (OPN), osteocalcin (OC)), (2) Change in the expression level of type I collagen protein, (3) Three items of alizarin red staining for directly evaluating calcification were evaluated.

(1) Evaluation of expression level of calcification marker gene Cells from each day are collected from dishes, and total RNA is extracted and purified using an RNA extraction and purification kit (SV Total RNA Isolation System, Promega). The expression level (mRNA level) of both osteopontin (OPN) and osteocalcin (OC) genes, which are generally used, was measured by quantitative PCR. At this time, cDNA used as a template for quantitative PCR was prepared by the reaction system shown in Table 1 below using an oligo dT20 primer. In addition, Ribatra Ace (Toyobo Co., Ltd.) was used as the reverse transcriptase.

  The reaction was performed at 42 ° C. for 1 hour using the above reaction system to prepare cDNA. Using this cDNA as a template, quantitative PCR was performed with the primer sets and annealing temperatures shown in Table 2 below. In addition, SYBR Green PCR Master Mix (made by Applied Biosystems) was used for the reaction of quantitative PCR, and reaction conditions followed the protocol of the said kit. As an internal control, the expression level of GAPDH gene in each sample was measured using GAPDH control reagents (Applied Biosystems). ABI Prizm 7000 sequence detection system (Applied Biosystems) was used for reaction and detection. The expression level in each sample was shown as a value (relative value) obtained by dividing the expression level of each gene by the expression level of the GAPDH gene.

  FIG. 1 shows time-dependent changes in the expression level of the osteopontin gene in odontoblasts and FIG. 2 shows the osteocalcin gene. As shown in FIG. 1, osteopontin gene expression was significantly induced by polyphosphate treatment (Example 1) from day 21 after calcification induction treatment, and was about 72 times that of no treatment (control) on day 21. It increased to about 120 times on the 35th day. Further, as shown in FIG. 2, the expression of osteocalcin gene was also induced by polyphosphate treatment (Example 1) after the 21st day after treatment, and peaks of increased expression were observed on the 21st and 35th days. It was. The expression level was about 18 times untreated (control) on day 21 and about 30 times on day 35. In the phosphoric acid treatment (Comparative Example 1), no increase in the expression level was observed as in the case of no treatment (control). In addition, compared to glycerophosphate + ascorbic acid treatment (Comparative Example 2), which is a positive control for cell calcification, the expression of polyphosphate treatment increased earlier for both genes, and the level of increased expression after the 21st day In total, polyphosphoric acid treatment was greater. From these results, it has been clarified that polyphosphate positively induces the expression of genes involved in cell calcification in odontoblasts and remarkably promotes calcification of odontoblasts.

(2) Expression of type I collagen protein The expression level of type I collagen protein in odontoblasts on the fifth day after the calcification induction treatment with each test substance was examined by immunostaining. The cells after the calcification induction treatment were fixed with 4% paraformaldehyde and then blocked with 5% skim milk. Subsequently, an antigen-antibody reaction was performed in the dark using a rabbit anti-human type I collagen antibody and a fluorescent (FITC) -labeled anti-rabbit IgG antibody. After nuclear staining using DAPI, cells were encapsulated using 0.1% PPDA solution, and expression of type I collagen protein was confirmed using a fluorescence microscope. Numerical analysis of the fluorescence intensity of FITC and DAPI after treatment with each test substance was performed using Image Gauge (Fuji Film Co., Ltd. image analysis software) based on the image of the microscope. Based on the digitized fluorescence intensity, the protein expression level per unit cell number was calculated as a relative value, and the type I collagen protein expression level by each treatment was compared. The results are shown in FIG. In polyphosphoric acid treatment (Example), the amount of expression increased compared to any of untreated (control), phosphoric acid treatment (Comparative Example 1), and glycerophosphoric acid + ascorbic acid treatment (Comparative Example 2). In particular, the amount of expression of type I collagen protein was approximately doubled in the treatment with polyphosphoric acid as compared with the case without treatment. From this, it was shown that polyphosphate promotes the expression of type I collagen.

(3) Evaluation of calcification by alizarin red staining The state of odontoblast calcification on the 29th day after the calcification induction treatment with each test substance was observed by alizarin red staining. After fixing the cells with 10% phosphate buffered formalin, calcium phosphate formed during calcification was stained with alizarin red, and the stained image was observed with a microscope. The results are shown in FIG. In no treatment (control) and phosphoric acid treatment (Comparative Example 1), no orange staining image showing the formation of calcified matter was observed, and no cellular calcification occurred. On the other hand, a remarkable stained image was observed in the polyphosphoric acid treatment (Example 1). From this, it was confirmed that cell calcification was induced by polyphosphoric acid.

(Test Example 2) Formation promotion of secondary dentin by polyphosphoric acid For the purpose of confirming the effect of promoting the formation of dentin in vivo by polyphosphoric acid, secondary dentin formation in an artificial degeneration cavity using rats An experiment was conducted to observe the above. A 6-week-old Wister rat is anesthetized, and a Class I cavity is formed on the occlusal surface of the maxillary first molar using a 1/4 round bar, and then a point-exposed medulla is formed using a probe to form an exposed cavity. did. The formed cavernous cavities were washed alternately with hypochlorous acid and hydrogen peroxide, then washed with physiological saline, and dried with a sterile cotton ball. After confirming the hemostasis, each of the cavities was coated with the preparation of the present invention prepared in Preparation Example 1 or a calcium hydroxide preparation (Daikar; manufactured by Dentsu Ply Sankin), which is generally used directly as a capping agent for comparison. Temporarily sealed with Fuji III (manufactured by GC).

  Rats that have been treated for a certain period after treatment are euthanized by inhalation anesthesia (ether), the maxilla is cut, and the tissue is fixed for 1 day by immersion fixation using 10% neutral buffered formalin solution (pH 7.4). went. Thereafter, acid decalcification was performed at room temperature for about 2 days. After the decalcification, the specimen was trimmed by excision with the second molar, and embedded in paraffin with the split surface down. Tissue sections were prepared and observed by HE staining.

  FIG. 5 shows stained images extracted from tissue specimens on the third day after each treatment of sodium polyphosphate (the preparation of the present invention) and calcium hydroxide (comparative preparation) (in the figure, the region surrounded by a circle is the treatment site) The arrow indicates the necrotic site of the pulp, D is dentin, DP is pulp, and AB is alveolar bone). In the treatment with calcium hydroxide, a necrotic layer was observed immediately below the coated material, followed by inflammatory cell infiltration mainly composed of neutrophils and dilation of blood vessels. In contrast, with sodium polyphosphate treatment, there was almost no necrotic layer in the dental pulp, and infiltration of inflammatory cells was limited to the very shallow part. This shows that the healing of the damaged site was promoted by polyphosphoric acid.

  FIG. 6A shows a stained image extracted from a tissue specimen on the seventh day after treatment with sodium polyphosphate (in FIG. 6A, the site surrounded by a circle indicates the treatment site and its periphery, and the site surrounded by a square is a secondary site. (D is the dentin and DP is the pulp). In addition, FIGS. 6B and 6C show enlarged views of the stained images (in FIG. 6C, ▲ indicates the formed dentin bridge).

  With calcium hydroxide treatment, inflammatory cell infiltration into the dental pulp continued, and tissue repair in the damaged area was hardly observed. In contrast, the treatment with sodium polyphosphate completely eliminated the inflammatory reaction, the tissue repair in the pulp progressed, and the appearance of bone-like dentin on the exposed pulp surface was observed. In addition, the formation of dentin bridge composed of dentin with bone-like dentin and dentinal tubule structure was observed, and it was confirmed that the formation of secondary dentin was promoted by polyphosphoric acid.

(Production Example) Production of polyphosphoric acid (medium-long chain polyphosphoric acid) 20 g of sodium hexametaphosphate of food additive standard was dissolved in 200 ml of purified water, and 32 ml of 96% ethanol was gradually added thereto. This was stirred well and allowed to stand at room temperature for about 30 minutes, followed by centrifugation (10,000 × g, 20 minutes, 25 ° C.) to separate the aqueous solution component and the precipitate. The aqueous solution component was discarded, and the collected precipitate was washed with 70% ethanol and dried in vacuo. In this way, 9.2 g of medium-long chain (average chain length of 60 or more) polyphosphate was obtained as a precipitate (yield 46.0%).

(Formulation example)
Sodium polyphosphate (average chain length 60) obtained according to the production example was mixed with 4% gelatin (made by Nitta Gelatin) at a concentration of 1% to obtain a gel-like dentin-forming pulp capping agent.

The time-dependent change of the expression level in the odontoblast of an osteopontin gene is shown. The time-dependent change of the expression level in the odontoblast of an osteocalcin gene is shown. 1 shows the expression level of type I collagen protein in odontoblasts. The result of investigating the state of odontoblast calcification by alizarin red staining is shown. The stained image extracted from the tissue specimen on the third day after each treatment with sodium polyphosphate and calcium hydroxide is shown. The stained image extracted from the tissue specimen on the 7th day after treatment with sodium polyphosphate and an enlarged view thereof are shown.

Claims (2)

The following general formula (I):
H _{n + 2} (P _n O _{3n + 1} ) (I)
(Wherein n represents an integer of 40 to 150) . A dentin-forming pulp capping agent containing polyphosphoric acid composed of one or a mixture of two or more linear phosphoric acids as an active ingredient. The dentine-forming pulp capping agent according to claim 1 , wherein the polyphosphoric acid is a polyphosphate.

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