Dentin bonding composition and pulp capping composition containing CPNE7-derived peptides

Abstract

The dentin bonding composition according to the present invention comprises a peptide having an amino acid sequence represented by the following general formula 1: KY-R1-R2-R3-R4-R5-R6-R7-R8 (General Formula 1). In General Formula 1, R1 is arginine (R), lysine (K), or glutamine (Q), R2 is arginine (R) or glutamine (Q), R3, R4, and R5 are arginine (R) or lysine (K), R6 is asparagine (N) or serine (S), and R7 and R8 are lysine (K) or tyrosine (Y). The composition according to the present invention can be used to treat damaged tooth structure such as caries or fractures, and can prevent pulp necrosis, tooth loss, or tooth loss that may occur after treatment.

Description

Technical Field

[0001] The present invention relates to a dentin adhesive and a composition for pulp capping, and more particularly, to a dentin adhesive and a composition for pulp capping containing a CPNE7-derived peptide that forms tubular dentin having a continuous structure with biocompatible tubular dentin.

Background Art

[0002] Various dental restorative materials have different physical properties (such as coefficient of thermal expansion, strength, wear degree, elastic modulus, etc.) from those of the tooth itself, and as a process for physically and chemically adapting the material to the tooth, processes such as tooth removal and chemical treatment of the tooth are involved. Chemical irritation, microleakage, and irritation during the treatment process of dental materials for preservation and prosthetic restoration can weaken or cause hypersensitivity and pulp lesions in existing pathological tooth problems (such as fracture, hypersensitivity, caries, etc.). This leads to necrosis of the pulp, the living tissue inside the tooth, and results in root canal treatment to remove the pulp, additional tooth removal, and defects. Also, when the dentin of the tooth is damaged due to various causes and the dentinal tubules are exposed to the outside, symptoms of tooth sensitivity will occur. Such symptoms may also occur when tooth substance is removed during the dental treatment process.

[0003] To reduce hypersensitivity symptoms caused by exposed dentinal tubules, dentinal tubule occlusion and physiological calcification are necessary. In order to overcome such limitations, the concept of physiological dentin sealing has been introduced, and there is a need for a new restorative material having a physiological activity that promotes the recovery of odontoblasts responsible for dentin secretion.

[0004] Recently, geriatric dental diseases associated with aging, which have emerged as a serious social problem, are also affecting the dental field due to the expanded application of national health insurance for the elderly population. The number of natural teeth in the elderly shows a tendency to decrease as the age increases, and there are various research results indicating that the lower the number of natural teeth, the lower the quality of life. Therefore, the demand for surgical procedures and materials that can preserve the patient's natural teeth as much as possible is increasing.

[0005] A tooth consists of hard tissues: enamel, dentin, and chalk, and the soft tissue inside, the pulp. The pulp has well-developed nerves and blood vessels, and odontoblasts, which form the dentin, are located at the boundary between the pulp and dentin. When the pulp is damaged for various reasons, such as caries or fracture, the symptoms experienced by the patient and the necessary treatment procedures will differ depending on the extent of the damage. If the tooth damage is limited to the enamel or dentin, the weakened tooth structure will be removed and then filled with restorative material. If the damage is close to the pulp or invades the pulp, and the exposed area of ​​pulp is small, the pulp will be directly sealed using a pulp capping material and the tooth will be restored.

[0006] To restore the structure and function of teeth after damage, various restorative and prosthetic materials are used, consisting of components such as amalgam, glass ionomer cement, composite resin, gold, and porcelain. Of these, resin is currently the most commonly used material for direct restoration, and dentin adhesive is used to bond the resin to the tooth structure. Currently, eighth-generation adhesives that shorten the application step are the most common, and a representative example of eighth-generation adhesives is Bisco Dental's All-Bond Universal product. If adhesives are developed that restore the thickness of damaged dentin, prevent microleakage, and have biological effects that induce the restoration of damaged dentin thickness when restoring with resin, it will be useful for protecting natural teeth in the future, which will improve overall oral health and quality of life.

[0007] When a small area of ​​pulp is exposed, or when the remaining tooth structure is thin and close to the pulp, pulp capping is applied to preserve the vitality of the pulp. An ideal pulp capping material does not cause inflammation of the pulp, bonds with the tooth structure, has no microleakage, forms a dentin bridge, and is clinically easy to use. Many materials, such as calcium hydroxide, MTA (Mineral Trioxide Aggregate), and adhesive resins, have been studied for successful pulp capping, but among these, MTA is used in the most diverse range of applications due to its biocompatibility. One of the important functions of direct pulp capping materials is their biological effect of stimulating odontoblasts within the pulp to induce new dentin formation. Such an effect allows for sealing of the exposed pulp and prevents the need for root canal treatment. Root canal treatment removes all of the pulp inside the tooth, making it difficult to preserve the natural tooth. Therefore, the development of direct pulp capping materials with dentin formation-inducing functions is also necessary for the overall improvement of oral health.

[0008] While resin restorative materials are commonly used, they offer good adhesion to tooth structure, but the toxicity of the monomers often causes patients to experience unpleasant symptoms after restoration. To minimize side effects associated with treatment, it is necessary to seal exposed dentinal tubules, restore the thickness of damaged dentin, and minimize the impact of monomers on the pulp. MTA, which is widely used as a pulp capping material, also has good biocompatibility and sealing ability, but it forms a restorative dentin that is different from the existing tubular dentin. In the long term, this restorative dentin can experience microscopic leakage at the boundary with the existing dentin. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The present invention aims to provide a dentin adhesive that prevents pulp necrosis, tooth removal, or tooth loss, which can occur when various dental restorative materials are applied to teeth.

[0010] Another objective of the present invention is to provide a pulp covering composition that can be directly applied to seal and repair the pulp when the extent of tooth damage is close to or invades the pulp, and the area of ​​exposed pulp is small.

[0011] The objects of the present invention are not limited to those mentioned above, and any other objects not mentioned should be clearly understood by a person with ordinary skill in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0012] According to one aspect of the present invention for solving the aforementioned technical problems, a dentin bonding composition is provided that contains a peptide having the amino acid sequence of the following general formula 1. KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the above general formula 1, R1 is arginine (R), lysine (K), or glutamine (Q). R2 is either arginine (R) or glutamine (Q), R3, R4, and R5 are arginine (R) or lysine (K), respectively. R6 is either asparagine (N) or serine (S). R7 and R8 are either lysine (K) or tyrosine (Y).

[0013] With regard to dentin adhesion, mutant peptides having a sequence that differs from the constituent amino acid sequence by one or more amino acid residues are also included in the category of peptides provided in this invention, as long as they can demonstrate an effect that can promote the physiological calcification of dentin.

[0014] In general, amino acid exchanges in proteins and polypeptides that do not alter the overall molecular activity are well known in the field. The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. These may also include peptides in which mutations or modifications in the amino acid sequence increase the structural stability of the peptide to heat, pH, etc., or increase its ability to promote dentin synthesis.

[0015] For example, glutamine, the acidic amino acid located at the third position of the peptide of SEQ ID NO: 1 provided in the present invention, can be replaced with lysine or arginine, both basic amino acids, while still exhibiting the effects of the peptide provided in the present invention; arginine, the basic amino acid located at the fourth or fifth position of the peptide of SEQ ID NO: 1, can be replaced with glutamine, an acidic amino acid, or lysine, a basic amino acid, while still exhibiting the effects of the peptide provided in the present invention; lysine, the basic amino acid located at the sixth, seventh, or ninth position of the peptide of SEQ ID NO: 1, can be replaced with arginine, a basic amino acid, or tyrosine, an aromatic amino acid, while still exhibiting the effects of the peptide provided in the present invention; asparagine, the acidic amino acid located at the eighth position of the peptide of SEQ ID NO: 1, can be replaced with serine, a neutral amino acid, while still exhibiting the effects of the peptide provided in the present invention; and tyrosine, the aromatic amino acid located at the tenth position of the peptide of SEQ ID NO: 1, can be replaced with lysine, a basic amino acid, while still exhibiting the effects of the peptide provided in the present invention.

[0016] Thus, even if the acidic amino acid, basic amino acid, or aromatic amino acid constituting the peptide of the present invention is replaced with a different acidic amino acid, basic amino acid, neutral amino acid, or aromatic amino acid, respectively, the peptide provided by the present invention can still exhibit the same effects. Therefore, it is obvious that mutant peptides having an amino acid sequence different from that of the amino acid sequence constituting the peptide of the present invention by one or more amino acid residues are also included in the scope of the peptides provided by the present invention.

[0017] In addition, even if the peptide of the present invention has a form in which an arbitrary amino acid is added to its N-terminus or C-terminus, it can still exhibit the same effects as the peptide provided by the present invention. Therefore, it is included in the scope of the peptides provided by the present invention. As an example, it may be in a form in which 1 to 300 amino acids are added to the N-terminus or C-terminus of the peptide. As another example, it may be in a form in which 1 to 100 amino acids are added to the N-terminus or C-terminus of the peptide. As still another example, it may be in a form in which 1 to 24 amino acids are added to the N-terminus or C-terminus of the peptide.

[0018] Specifically, the peptide according to General Formula 1 can be represented by Tables 1 to 12 below.

[0019] For example, the peptide (SEQ ID NO: 1) can be synthesized by the Fmoc (9-fluorenylmethyloxycarbonyl) method, and the amino acids of the synthesized peptide can be substituted to synthesize the peptides of each group (Tables 1 to 12). N-KYQRRKKNKY-C (SEQ ID NO: 1)

[0020] First, the peptides of Group 1 can be synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 or the peptide of SEQ ID NO: 1 with lysine or arginine (Table 1).

[0021] [Table 1]

[0022] Next, the peptide of Group 2 can be synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 with lysine or arginine, and substituting the 8th amino acid with serine (Table 2).

[0023] [Table 2]

[0024] Next, the peptide of Group 3 can be synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 with lysine or arginine, substituting the 9th amino acid with tyrosine, and substituting the 10th amino acid with lysine (Table 3).

[0025] [Table 3]

[0026] Next, the peptide of Group 4 can be synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 with lysine or arginine, substituting the 8th amino acid with serine, substituting the 9th amino acid with tyrosine, and substituting the 10th amino acid with lysine (Table 4).

[0027] [Table 4]

[0028] Next, the peptide of Group 5 can be synthesized by substituting the 3rd amino acid of the peptide of SEQ ID NO: 1 with arginine, substituting the 4th amino acid with glutamine, and substituting the 5th to 7th amino acids with lysine or arginine (Table 5).

[0029] [Table 5]

[0030] Next, the peptides of group 6 can be synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with arginine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, and the eighth amino acid with serine (Table 6).

[0031] [Table 6]

[0032] Next, the peptides of group 7 can be synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with arginine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 7).

[0033] [Table 7]

[0034] Next, the peptides of group 8 can be synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with arginine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the eighth amino acid with serine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 8).

[0035] [Table 8]

[0036] Next, the peptides of group 9 can be synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, and the fifth to seventh amino acids with lysine or arginine (Table 9).

[0037] [Table 9]

[0038] Next, the peptides of group 10 can be synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, and the eighth amino acid with serine (Table 10).

[0039] [Table 10]

[0040] Next, the peptides of group 11 can be synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 11).

[0041] [Table 11]

[0042] Finally, the peptides of group 12 can be synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the eighth amino acid with serine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 12).

[0043] [Table 12]

[0044] In another embodiment, the present invention provides a polynucleotide encoding the peptide.

[0045] The polynucleotide may be mutated by substitution, deletion, insertion, or combination thereof of one or more bases. When the nucleotide sequence is produced by chemical synthesis, synthetic methods known in the art, such as those described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), can be used, and synthesis can be carried out using phosphotriester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis on solid supports, etc. For example, the polynucleotide encoding the peptide of the present invention may contain the base sequence of SEQ ID NO: 4.

[0046] In another embodiment, the present invention provides an expression vector containing the polynucleotide, a transformant containing the expression vector, and a method for producing the peptide using the transformant.

[0047] In this invention, the term "expression vector" refers to a recombinant vector capable of expressing a target peptide in target host cells, and is a gene crop containing essential regulatory elements operably linked to the expression of a gene insert. The expression vector includes expression regulatory elements such as a start codon, a termination codon, a promoter, and an operator, wherein the start and termination codons are generally considered to be part of the nucleotide sequence encoding the polypeptide, and must exert their effect in the organism when the gene crop is administered, and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inductive.

[0048] In this invention, the term "operably linked" refers to a state in which a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to influence the expression of the coding sequence. Operational linking with an expression vector can be produced using genetic engineering techniques known in the art, and site-specific DNA cleavage and linking can be performed using enzymes or other methods commonly known in the art.

[0049] Furthermore, the expression vector may include a signal sequence for peptide efflux to facilitate the separation of the peptide from the cell culture medium. Specific start signals may also be necessary for efficient translation of the inserted nucleic acid sequence. These signals include an ATG start codon and adjacent sequences. In some cases, an exogenous translational regulatory signal, which may include an ATG start codon, may be required. These exogenous translational regulatory signals and start codons can come from a variety of natural and synthetic sources. Expression efficiency may be increased by introducing appropriate transcription or translational enhancers.

[0050] In addition, the expression vector may further include a protein tag that can be optionally removed using an endopeptidase to facilitate the detection of the peptide.

[0051] In this invention, the term "tag" refers to a molecule that exhibits quantifiable activity or properties, and may be a fluorescent molecule containing a chemical fluorescent substance (fluoracer) such as fluorescein, a polypeptide fluorescent substance such as green fluorescent protein (GFP) or an associated protein, or an epitope tag such as a Myc tag, Flag tag, histidine tag, leucine tag, IgG tag, or streptavidin tag. In particular, when using an epitope tag, a peptide tag consisting of 6 or more amino acid residues is preferably used, and more preferably 8 to 50 amino acid residues can be used.

[0052] In the present invention, the expression vector may include a nucleotide sequence encoding a peptide that provides the effect of promoting the physiological calcification of dentin according to the present invention, but the vector used is not particularly limited as long as it can produce the peptide, but preferably it is plasmid DNA, phage DNA, etc., and more preferably commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), plasmids derived from Escherichia coli (pYG601BR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (pUB110, pTP5, etc.), plasmids derived from yeast (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retrovirus, adenovirus, vaccinia virus, etc.) These can be insect virus vectors (such as baculoviruses), etc. Since the expression levels and modifications of the aforementioned expression vectors vary depending on the host cell, it is desirable to select and use the host cell that best suits the purpose.

[0053] The transformants provided in the present invention are produced by introducing the expression vector provided in the present invention into a host and performing transformation, and can be used to express the polynucleotides contained in the expression vector and produce the peptide. The transformation can be carried out by various methods, but is not particularly limited as long as the peptide can be produced. Possible methods include CaCl2 precipitation, the Hanahan method which improves efficiency by using a reducing agent called DMSO (dimethyl sulfoxide) in addition to CaCl2 precipitation, electroporation, calcium phosphate precipitation, plasmofusion, stirring method using silicon carbide fibers, agrobacteria-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation. Furthermore, the host used to produce the transformants is not particularly limited as long as it can produce the peptide, but may be bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; yeast cells such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; fungal cells such as Pichia pastoris; insect cells such as Drosophila and Spodoptera frugiperda Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells; or plant cells.

[0054] The transformant may also be used in a method for producing a peptide that provides the effect of promoting the physiological calcification of dentin according to the present invention. Specifically, a method for producing a peptide that provides the effect of promoting the physiological calcification of dentin according to the present invention may include (a) culturing the transformant to obtain a culture, and (b) recovering the peptide of the present invention from the culture.

[0055] In this invention, the term "culture" refers to a method of growing microorganisms under appropriately artificially controlled environmental conditions. In this invention, the method of culturing the transformants can be carried out by methods known in the art. Specifically, the culture is not particularly limited as long as it can be produced by expressing the peptide that provides the effect of promoting the physiological calcification of dentin in this invention, but it can be carried out continuously by batch processing, fed-batch culture, or repeated fed-batch process.

[0056] The culture medium used for cultivation must meet the requirements of the specific strain by adjusting the temperature, pH, etc., under aerobic conditions within a standard culture medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc., using appropriate methods. Possible carbon sources include a mixture of glucose and xylose as the main carbon source, as well as sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used individually or in mixtures. Furthermore, possible nitrogen sources include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids and peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their degradation products, defatted soy cake or its degradation products. These nitrogen sources may be used individually or in combination. The culture medium may contain monopotassium phosphate, dispotassium phosphate, and corresponding sodium-containing salts as phosphorus sources. Possible phosphorus sources include potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or corresponding sodium-containing salts. Inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used. Finally, in addition to the above substances, growth-essential substances such as amino acids and vitamins may be used.

[0057] Furthermore, appropriate precursors may be used in the culture medium. The aforementioned raw materials may be added to the culture in batch, fed-batch, or continuous mode by an appropriate method during the culture process, but are not limited to these methods. The pH of the culture may be adjusted by using basic compounds such as sodium hydroxide, potassium hydroxide, or ammonia, or acidic compounds such as phosphoric acid or sulfuric acid by an appropriate method.

[0058] Furthermore, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble formation. To maintain aerobic conditions, oxygen or oxygen-containing gas (e.g., air) is injected into the culture medium. The culture temperature is typically 27°C to 37°C, preferably 30°C to 35°C. Culturing is continued until the maximum amount of peptide is produced. For this purpose, this is usually achieved in 10 to 100 hours.

[0059] Furthermore, the step of recovering the peptide from the culture can be carried out by methods known to the art. Specifically, the recovery method is not limited to these, as long as it can be used to recover the produced peptide, but preferably methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), and chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used.

[0060] In this invention, the term "prevention" means any action by which the administration of the dentin bonding composition and pulp capping composition containing the peptide of the present invention inhibits or delays the occurrence of pulp necrosis, tooth removal or tooth loss, or secondary caries or pulpitis that may occur due to dental restorative materials.

[0061] In this invention, the term "treatment" means any act that enables dental restorative treatment by administering a restorative material, a dentin adhesive composition, or a pulp capping composition containing the peptide of the present invention as an active ingredient to an individual requiring dental restorative treatment, thereby promoting physiological remineralization of dentin between the tooth and the restorative material.

[0062] The compositions of the present invention may be prepared in the form of compositions for treating damaged tooth structure, such as caries or fracture, further comprising a suitable carrier (natural or non-natural carrier), excipient, or diluent commonly used in the preparation of the aforementioned tooth restorative compositions. Specifically, each of the compositions may be formulated in the form of a sterile injectable solution that can be administered by conventional methods to the site where tooth structure damage, such as caries or fracture, has been induced. Examples of carriers, excipients, and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and collagen. When formulated, these can be prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. In particular, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories, and ointments (e.g., pulp liners) may be used. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol (registered trademark), macrogol (registered trademark), tween (registered trademark) 61, cocoa butter, lauric acid butter, and glycerol gelatin.

[0063] The content of the peptide in the composition of the present invention is not particularly limited, but may be 0.0001 to 50% by weight, more preferably 0.01 to 20% by weight, based on the total weight of the final composition.

[0064] The compositions of the present invention may be administered in pharmaceutically effective amounts, where the term “pharmaceutically effective amount” in the present invention means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level may be determined based on the severity of the disease, the activity of the drug, the patient’s age, weight, health status, sex, the patient’s sensitivity to the drug, the time of administration, route of administration, and elimination ratio of the compositions of the present invention used, the duration of treatment, the drugs used in combination with or concurrently with the compositions of the present invention, and other factors known in the medical field. The compositions of the present invention may be administered alone or in combination with known compositions for treating damaged tooth structure, such as caries or fractures. Taking all of the above factors into consideration, it is important to administer an amount that provides the greatest effect with the minimum amount without side effects.

[0065] The dosage of the composition of the present invention can be determined by those skilled in the art, taking into consideration the intended use, the severity of the disease, the patient's age, weight, sex, medical history, or the type of substance used as the active ingredient. For example, the composition of the present invention can be administered to an adult at a dose of about 0.1 ng / kg to about 100 mg / kg, preferably 1 ng / kg to about 10 mg / kg. The frequency of administration of the composition of the present invention is not particularly limited, but it may be administered once a day or divided into doses and administered several times a day. The aforementioned dosage does not limit the scope of the present invention in any way.

[0066] In another aspect, the present invention provides a method for treating damaged tooth structure, such as caries or fracture, which includes the step of administering the composition in a pharmaceutically effective amount to an individual who has developed damaged tooth structure, such as caries or fracture.

[0067] The term "individual" in this invention may include, without limitation, mammals such as rats and livestock that require treatment for damaged tooth structure such as caries or fracture, but humans may be excluded from among individuals that have developed the aforementioned disease.

[0068] The therapeutic compositions for caries, fracture, and other damaged tooth structure of the present invention may be administered via any common route, as long as they can reach the target tissue. The compositions of the present invention may be provided in any dosage form suitable for topical application, depending on the purpose, but are not limited thereto. For example, they may be administered orally, transdermally, intravenously, intramuscularly, or by subcutaneous injection. The compositions may be, but are not limited thereto, injectable, topical solutions, suspensions, emulsions, gels, puffs, or sprays. The dosage forms can be readily manufactured according to common methods of the art, and may use surfactants, excipients, hydrates, emulsifiers, suspensions, salts or buffers for osmotic pressure adjustment, colorants, spices, stabilizers, preservatives, or other commonly used adjuvants as appropriate.

[0069] In another embodiment, the present invention provides a quasi-drug composition for preventing or improving damaged tooth structure, such as caries or fracture, which comprises the peptide.

[0070] In this invention, the term "improvement" means all actions that at least reduce parameters related to the condition being treated, such as the severity of symptoms.

[0071] In the present invention, the term "improvement" can be interpreted as meaning all actions that involve administering a composition containing the peptide of the present invention as an active ingredient to an individual who requires treatment for damaged tooth structure such as caries or fracture, thereby promoting dentin synthesis and improving or benefiting from the symptoms of damaged tooth structure such as caries or fracture.

[0072] In this invention, the term "quasi-drug" refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans or animals, which have a milder effect than pharmaceuticals. For example, according to the Pharmaceutical Affairs Law, quasi-drugs are articles excluding those used for pharmaceutical purposes, and include textile and rubber products used for the treatment or prevention of diseases in humans and animals, items that have a mild or no direct effect on the human body and are not instruments or machines, as well as similar items, and disinfectants and insecticides for preventing infectious diseases.

[0073] In the present invention, the type and dosage form of the quasi-drug composition containing the peptide are not particularly limited, but examples include disinfectant and cleansing agents for skin or hair, cleansing products for skin or hair, soaps, shampoos, and skin ointments.

[0074] In another embodiment, the present invention provides a health functional food composition comprising the peptide for preventing or improving damaged tooth structure such as caries or fracture.

[0075] In this invention, the term "food" includes all foods in the ordinary sense, such as meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen noodles, other noodle products, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods.

[0076] The term "functional food" refers to a food with high medical and therapeutic effects, processed to efficiently exhibit biological regulatory functions in addition to nutrient supply. "Functionality" means regulating nutrients or obtaining effects useful for health purposes, such as physiological effects, on the structure and function of the human body. The food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and components commonly used in the industry. Furthermore, the dosage form of the food can also be manufactured without restriction, as long as it is a dosage form recognized as a food. The food composition of the present invention can be manufactured in various dosage forms, and unlike general pharmaceuticals, it has the advantage of using food as a raw material, thus avoiding side effects that can occur with long-term use of pharmaceuticals. It is also highly portable, and the food of the present invention can be taken as an adjunct to enhance the effect of preventing or improving damaged tooth structure, such as caries and fractures.

[0077] The term "health food" refers to foods that have a more active effect on maintaining or promoting health compared to general foods, while "health supplement food" refers to foods intended for health supplementation. In some cases, the terms "health functional food," "health food," and "health supplement food" may be used interchangeably.

[0078] Specifically, the aforementioned health functional foods are foods in which the peptide of the present invention is added to food ingredients such as beverages, teas, spices, gums, and confectionery, or manufactured as encapsulated, powdered, or suspended, and which, when consumed, produce specific health effects. However, unlike general pharmaceuticals, they have the advantage of being made from food ingredients and not having the side effects that can occur with long-term use of pharmaceuticals.

[0079] Since the food composition of the present invention can be consumed on a daily basis, it can be expected to have a high effect in preventing or improving damaged tooth structure such as caries and fractures, and therefore can be used very effectively.

[0080] The food composition may further contain a physiologically acceptable carrier, but the type of carrier is not particularly limited, and any carrier commonly used in the art may be used.

[0081] Furthermore, the food composition may contain additional ingredients commonly used in food compositions that can improve smell, taste, appearance, etc. For example, it may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. It may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr). It may also contain amino acids such as lysine, tryptophan, cysteine, and valine.

[0082] Furthermore, the food composition may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder and high-grade bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color fixatives (sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (monosodium glutamate (MSG), etc.), artificial sweeteners (dulcin, cyclamic acid, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum (potassium aluminum sulfate), potassium bitartrate, etc.), fortifiers, emulsifiers, thickeners, coating agents, gum bases, defoamers, solvents, and improvers. The additives may be selected according to the type of food and used in appropriate amounts.

[0083] The peptide of the present invention can be added as is or used in combination with other foods or food components, and can be used as appropriate in the usual manner. The amount of active ingredient mixed can be suitably determined according to its intended use (prevention, health, or therapeutic treatment). In general, when manufacturing food or beverages, the food composition of the present invention can be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, per 100 parts by weight of the food or beverage composition. However, for long-term intake for health and hygiene purposes, the active ingredient may be included in amounts below the above range, and there are no safety concerns, so it can also be used in amounts above the above range.

[0084] One example of the food composition of the present invention may be used as a health beverage composition, in which case, like ordinary beverages, it may contain various flavorings or natural carbohydrates as additional ingredients. The aforementioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame. The ratio of the aforementioned natural carbohydrates is usually about 0.01g to 0.04g per 100mL of the health beverage composition of the present invention, specifically about 0.02g to 0.03g.

[0085] In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavorings, colorings, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. It may also contain fruit pulp for the production of natural fruit juices, fruit juice beverages, or vegetable beverages. Such components may be used individually or in combination. While the proportion of such additives is not particularly important, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

[0086] The food composition of the present invention may contain various weight percentages if it can demonstrate a preventive or ameliorative effect on damaged tooth structure, such as caries or fracture. Specifically, the peptide of the present invention may be contained in an amount of 0.00001 to 100% by weight or 0.01 to 80% by weight relative to the total weight of the food composition, but is not limited thereto. [Effects of the Invention]

[0087] The dentin adhesive according to the embodiment of the present invention can prevent or suppress secondary caries or pulpitis that may occur due to dental restorative materials by preventing or preventing pulp necrosis, tooth removal, or tooth loss that may occur when various dental restorative materials are applied to teeth.

[0088] The pulp capping composition according to the embodiment of the present invention can be applied directly to seal and repair the pulp when the extent of tooth damage is close to or invades the pulp, and when the area of ​​exposed pulp is small. This enables pulp sealing, prevents or suppresses micro-leakage that may occur at the boundary of the repaired dentin, and provides high reliability and sustainability for the treatment of tooth damage.

[0089] The effects of the present invention are not limited to those mentioned above, and any other effects not mentioned will be clearly understood by a person with ordinary skill in the art to which the present invention pertains from the following description. [Brief explanation of the drawing]

[0090] [Figure 1] Figure 1 shows the effect of a mixed solution of dentin adhesive and KH001 on the expression of odontogenic differentiation marker genes in human dental pulp cells. The marker genes were DSPP, DMP1, and BSP, and gene expression was measured by RT-PCR. All values ​​in the results are shown as the mean and standard deviation of the results from three experiments, and *p<0.05 indicates a comparison with the control group. [Figure 2] Figure 2 shows the results of measuring dentinal tubular permeability when a mixed solution of dentin adhesive and KH001 was applied to human teeth with exposed dentin, to determine whether KH001 successfully reached the pulp. A is a schematic diagram of the sample preparation process for measuring dentinal tubular permeability, and B-D are the results captured using a confocal microscope. [Figure 3]Figure 3 shows the results of a histological evaluation of the tubular dentin regeneration capacity of a mixed solution of dentin adhesive and KH001. After exposing the dentin by making a saucer-shaped injury at the cervical region of Beagle dog teeth, the control group was treated with dentin adhesive without KH001 and then restored with resin, while the experimental group was treated with dentin adhesive containing KH001 and then restored with resin. Subsequently, histological evaluation was performed using H&E (Hematoxylin Eosin) staining to investigate the formation of tubular dentin. The dotted lines indicate the boundaries of newly formed tubular dentin, and TD (tertiary dentin) in the figure refers to tubular dentin. [Figure 4] Figure 4 shows the results of investigating the expression levels of odontogenic differentiation marker genes in human dental pulp cells at different KH001 concentrations under MTA-conditioned medium conditions. The marker genes included DSPP, DMP1, and BSP, and gene expression was measured by RT-PCR. All values ​​in the results are shown as the mean and standard deviation of the results from three experiments, with *p<0.05 and **p<0.01 indicating results compared to the control group. [Figure 5a] Figure 5a shows the results of examining changes in the expression of odontoblast differentiation gene markers on day 7 of differentiation in human dental pulp cell experiments treated with MTA-modified medium, in order to confirm the efficacy of KH001. [Figure 5b] Figure 5b shows the results of examining changes in the expression of odontoblast differentiation gene markers on day 14 of differentiation in human dental pulp cell experiments treated with MTA-modified medium, in order to confirm the efficacy of KH001. [Figure 6] Figure 6 shows the results of an experiment in which changes in odontoblast differentiation and calcification ability were examined in human dental pulp cells treated with MTA-modified medium to confirm the efficacy of KH001. [Figure 7] Figure 7 shows the results of examining changes in wound healing ability and cell migration ability in human dental pulp cell experiments treated with MTA-modified medium, in order to confirm the efficacy of KH001. [Figure 8]Figure 8 is a photograph taken with a confocal microscope to examine the permeability of the dentinal tubules of the pulp capping composition according to an embodiment of the present invention. [Figure 9] Figure 9 is a schematic diagram showing the application sites and the appearance before and after application of the dentin bonding composition and the pulp covering composition during the treatment of a damaged tooth. [Modes for carrying out the invention]

[0091] The objectives and effects of the present invention, as well as the technical configurations for achieving them, will become clear by referring to the embodiments described in detail below, along with the accompanying drawings. In the description of the present invention, if it is determined that a specific description of a known function or configuration would be unnecessary and obscure the gist of the invention, such detailed description will be omitted. Furthermore, the terms used below are defined for the purpose of describing the embodiments of the present invention and may change depending on the intent or conventions of the user or operator.

[0092] However, the present invention is not limited to the embodiments disclosed below and can be realized in a variety of different forms. These embodiments are provided solely to ensure the complete disclosure of the present invention and to fully inform those skilled in the art of the invention of the category of invention, which is defined only by the claims. Therefore, the definition should be based on the content throughout this specification.

[0093] The following describes specific embodiments of the present invention. (Example 1: Materials and Method) [Example 1-1: Peptide Preparation] The KH001 peptide (SEQ ID NO: 96) consists of a synthetic peptide corresponding to a fragment of the hCPNE7 protein, specifically amino acid residues 344-353 (KYKQKRRSYK). Peptide KH001 was synthesized using an Fmoc (9-fluorenylmethoxycarbonyl)-based solid-phase method, and its purity was measured by high-performance liquid chromatography to be over 97%.

[0094] [Examples 1-2: Cell Culture] MDPC-23 (mouse pre-odontoblast cell line) cells were provided by Dr. JENor (University of Michigan, Ann Arbor, MI, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL., Carlsbad, CA, USA). C3H10T1 / 2 cells were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and cultured in RPMI 1640 medium (Gibco BRL). Both cell lines were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL) and antibiotics / antifungal agents (Gibco BRL) at 37°C under a 5% CO2 atmosphere.

[0095] Cells were cultured in Minimum Essential Medium α (MEM-α; Gibco BRL) for use in in vitro and ex vivo experiments. For differentiation into human dental pulp cells (hDPC) and MDPC-23 cells, 80%–90% fused cells were cultured for 3 weeks in the appropriate medium supplemented with 5% FBS, ascorbic acid (50 μg / mL), and β-glycerophosphate (10 mM). Passages 2–4 were used for hDPSCs, and passages 23–25 were used for MDPC-23 cells.

[0096] (Example 2) [Example 2-1-1: Preparation of a dentin bonding composition] KH001 and Bisco Dental®'s dentin adhesive ABU (All-Bond Universal) were mixed by adding 2 mg of KH001 per 1 mL of ABU and stirring with a stirrer for approximately 40 minutes under reduced pressure (-750 mmHg) (stirring conditions: PADDLE 10-30 rpm, DISPERSE 500-600 rpm, HOMO 2400-3200 rpm). Afterwards, the mixture was stored in a light-shielded container. The prepared dentin adhesive composition was refrigerated at 4°C for approximately 3 months after preparation and applied to Examples 2-3 and 2-4, as shown in Table 13.

[0097] [Example 2-1-2: Preparation of a dentin bonding composition] In the cell experiments using human dental pulp cells (hDPC) prepared in Example 1-2, ABU was used to prepare the dentin adhesive composition by diluting it in 10% α-MEM at a ratio of 1:5000 to prevent side effects such as cell death due to the toxicity of the dentin adhesive monomer. KH001 peptide powder was dissolved in the aforementioned dilution to prepare compositions containing 5 ug / ml and 10 ug / ml, respectively, and the dentin adhesive compositions thus prepared were applied to Example 2-2. The prepared dentin adhesive compositions were stored refrigerated at 4°C after preparation.

[0098] [Example 2-2: Analysis of expression regulation of odontoblast differentiation marker genes] Human dental pulp cells (hDPCs) prepared in Example 1-2 were treated with the dentin bonding composition of Example 2-1-2, and then the expression of DSPP, DMP1, and BSP genes was measured by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analysis. The gene expression values ​​shown in Figure 1 are the mean and standard deviation of the results from three experiments, with *p<0.05 and **p<0.01 indicating results compared to the control group.

[0099] Referring to Figure 1, the expression levels of the odontoblast differentiation marker genes DSPP, BSP, and DMP1 increased as the concentration of KH001 increased. When treated with KH001 at a concentration of 10 ug / ml, the mRNA expression of DSPP, DMP1, and BSP increased by more than 2.5 times.

[0100] [Example 2-3: Observation of dentinal tubule permeability] The dentin bonding composition prepared according to Example 2-1-1 of the present invention was applied to extracted human teeth, and the dentinal tubular permeability was observed. The extracted human teeth were impacted human third molars from patients aged 18 to 22 years, provided by Seoul National University Dental Hospital. The experimental protocol was approved by the Institutional Review Board (IRB number: S-D20140007), and prior consent was obtained from all patients.

[0101] After exposing the dentinal tubules by cutting the crown portion of an extracted human tooth transversely with a diamond saw, a dentin bonding composition prepared according to Example 2-1-1, which contains KH001 peptide (SEQ ID NO: 96) with rhodamine as a fluorescent staining reagent, was applied to the crown surface of the extracted human tooth.

[0102] Figure 2 shows the results of confocal imaging after applying the dentin bonding composition without a brushing or air-drying process, allowing it to stand for approximately 30 seconds before photopolymerization.

[0103] Figure 2 shows the results of measuring dentinal tubular permeability when a mixed solution of dentin adhesive and KH001 was applied to a human tooth with exposed dentin for approximately 30 seconds, to determine if KH001 successfully reached the pulp. Referring to Figure 2, A is a schematic diagram of the sample preparation process for measuring dentinal tubular permeability. Figures B to D in Figure 2 were taken with a confocal microscope, and observations confirmed that the peptide KH001 successfully flowed along the dentinal tubules into the pulp cavity.

[0104] [Examples 2-4: Histological evaluation of tubular dentin regeneration capacity] After exposing the dentin by making a saucer-shaped injury at the cervical region of the teeth of Beagle dogs, the control group had dentin adhesive without peptide KH001 applied and then restored with resin, while the experimental group had dentin adhesive containing KH001 applied and then restored with resin. Subsequently, histological evaluation was performed by H&E staining to investigate the formation of tubular dentin.

[0105] Figure 3 shows the histological evaluation of the tubular dentin regeneration ability of the dentin bonding composition of Example 2-1-1. Referring to Figure 3, the dotted lines indicate the boundaries of newly formed tubular dentin, and TD (newly formed tertiary dentin) is dentin containing newly formed dentinal tubules.

[0106] Referring to Figure 3, in the control group (I) to which only dentin adhesive (ABU) was applied, tubular dentin was not formed below the site of dentin damage. However, in the test groups (II) (containing 5 ug / ml of KH001) and (III) (containing 10 ug / ml of KH001) to which the dentin adhesive composition prepared according to Example 2-1-2 was applied, it was confirmed that tubular dentin was formed on the pulp side below the site of dentin damage. Furthermore, odontoblasts were arranged in a surrounding pattern in the pulp adjacent to the newly formed tubular dentin, indicating that it was an activated, healthy pulp.

[0107] Table 13 shows the results of a shear bond strength test performed using the Ultradent method to confirm the adhesion strength of the dentin bonding composition containing KH001 prepared according to Example 2-1-1 to tooth structure. After preparing the dentin surface with 320-grit and 600-grit sandpaper, the control group (Bisco's ABU) and the experimental group (dentin adhesive containing KH001 at a concentration of 2 mg / mL) were applied using a self-etching method for approximately 10-15 seconds, and subjected to a 1000 mW / cm² test. 2After photopolymerization with an intensity light source, scrubbing was performed, and DLU (Duo-Link Universal®, Bisco) was bonded by irradiating with the same intensity light source for 20 seconds. The test specimens were then stored in 37°C pure water (DI water) before measurement. Each group contains 10 samples.

[0108] [Table 13]

[0109] Table 13 summarizes the adhesive strength of the dentin adhesive composition produced according to Example 2-1-1. As can be seen in Table 13, even though KH001 was included at a high concentration of approximately 200 times the effective concentration, the dentin adhesive composition provided an adhesive strength comparable to that of conventional dentin adhesives. These results suggest that even if KH001 is included in the dentin adhesive composition at a concentration of approximately 2 mg / mL or less, it can provide an adhesive strength equivalent to that of conventional dentin adhesives.

[0110] (Example 3-1: Preparation of a composition for pulp capping) MTA (ProRoot MTA; Dentsply Sirona, New York, PA, USA) was mixed on a sterile glass plate using a metal slab, according to the manufacturer's instructions regarding the powder / liquid ratio, and then cured at room temperature for 1 hour for complete hardening.

[0111] After hardening was complete, the MTA was crushed into a powder in a sterile mortar and pestle, immersed in MEM-α (Gibco® BRL), a culture medium for human dental pulp cells, for 24 hours, and then stored at approximately 37°C to prepare the conditioned media.

[0112] Subsequently, KH001 was treated with a prepared medium diluted to a ratio of x1 / 5 at concentrations of 5 ug / mL and 10 ug / mL, respectively, to prepare a pulp capping composition.

[0113] (Example 3-2: Evaluation of odontogenic differentiation marker genes (DSPP, DMP1, BSP) and CPNE7 gene expression levels at different concentrations of KH001 in a pulp capping composition) After extracting total RNA from human dental pulp cells using Tri-Reagent®, cDNA was synthesized by treating the cells with reverse transcriptase and oligo(dT) primers (Invitrogen).

[0114] For the gene amplification process, 1 μL of reverse-transcribed cDNA, primers corresponding to each gene, and SYBR® Green Supermix were mixed in a 96-well plate, and then a quick cycle was performed according to the manufacturer's instructions.

[0115] The amount of PCR results was measured as a ratio to GAPDH (glyceraldehyde 3-phosphate dehydrogenase), and the relative expression levels were compared using the comparative threshold cycle (CT) method.

[0116] Figure 4 shows the results of measuring the expression levels of odontogenic differentiation marker genes and the CPNE7 gene at different concentrations of the pulp capping composition KH001 (KH001 under MTA-modified medium conditions) in human dental pulp cells using RT-PCR. All values ​​are shown as the mean and standard deviation of the results from three experiments, with *p<0.05 and **p<0.01 indicating comparisons with the control group.

[0117] (Example 3-3: Evaluation of daily differentiation expression levels of odontoblast differentiation marker genes (DSPP, DMP1, BSP) and CPNE7 gene after treatment with a pulp capping composition (KH00110ug / mL under MTA-modified medium conditions)) To compare and measure the function of the pulp capping composition containing KH001 10ug / ml prepared according to Example 3-1 in inducing differentiation of human pulp cells into odontoblasts, a control group treated only with differentiation medium (Ctrl), a group treated with MTA-modified differentiation medium (MTA), and a group treated with the pulp capping composition (MTA-modified / CPNE7 functional peptide together, MTA+KHOO1) were compared.

[0118] The differentiation medium was basically prepared by adding 5% FBS to α-MEM, along with 1% antibiotic, 10 mM β-glycerophosphate, and 50 μg / mL ascorbic acid.

[0119] After treating human dental pulp cells with each culture medium, RT-PCR was performed on days 0, 7, and 14 to examine changes in the expression of odontoblast differentiation gene markers.

[0120] Figure 5 shows the results of examining changes in the expression of odontoblast differentiation gene markers on a day-by-day basis to confirm the efficacy of KH001 (MTA+KH001) in human dental pulp cell experiments treated with MTA-modified medium. MTA is a preparation applied when the dental pulp is exposed, or when the dental pulp is not exposed but only a thin layer of dentin remains and pulp damage is expected. Therefore, there is a high possibility that odontoblasts originally present in the exposed dental pulp area will die, and in order to restore this and form tertiary dentin in the exposed dental pulp area, it is important to have a function that induces the differentiation of dental pulp cells into odontoblasts.

[0121] Referring to Figure 5, when the expression of odontoblast markers and calcification-related genes was examined during the differentiation process of human dental pulp cells, the expression levels of DMP1 and CPNE7 were significantly increased in the group treated with both MTA and CPNE7 functional peptide on day 7 of differentiation compared to the group treated with MTA alone. On day 14, the expression of DSPP, DMP1, and CPNE7 genes was significantly higher in the group treated with both MTA and CPNE7 functional peptide than in the group treated with MTA alone. BSP is a gene involved in bone and dentin formation, and its expression was not significantly increased in the group treated with MTA alone on either day 7 or day 14, and was observed to be expressed to a similar degree as in the group treated with MTA alone.

[0122] (Examples 3-4: Evaluation of differentiation and calcification ability of odontoblasts treated with MTA-modified medium) Alizarin red staining (ARS) was performed to investigate the effects of CPNE7 functional peptide on the differentiation and calcification ability of odontoblasts in an MTA-treated environment.

[0123] Human dental pulp cells (passage 2-4) are placed in a 6-well plate in a 1 x 10⁶ arrangement. 5 After inoculation at a cell / well density, once a confluency of approximately 80% was reached, the differentiation medium corresponding to each control and experimental group was treated for 7, 14, and 21 days, with the differentiation medium being changed every two days.

[0124] To fix the cells, they were treated with 4% paraformaldehyde at 4°C for 15 minutes, and the calcified cells were stained with 40 mM Alizarin Red S using the ARed-Q kit (Alizarin Red S staining Quantification kit) at room temperature for 30 minutes.

[0125] Subsequently, for quantitative analysis, the solution was treated with 10% acetic acid at room temperature for 30 minutes, then centrifuged to extract 500 μL of the upper layer, and 200 μL of 10% ammonium hydroxide was added for neutralization.

[0126] Subsequently, absorbance was measured at 405 nm along with the ARS standard, and all experiments were performed in three sets.

[0127] Figure 6 shows the results of an experiment in which changes in odontoblast differentiation and calcification ability were examined in human dental pulp cells treated with MTA-modified medium to confirm the efficacy of KH001 (composition for dental pulp covering).

[0128] The clinical situations in which MTA is applied are those where the dental pulp is exposed and odontoblasts have died. In order to maintain the dental pulp in such situations, it is necessary to induce differentiation of pulp cells into odontoblasts, and for the differentiated odontoblasts to effectively secrete calcifying substances. Through this process, the exposed pulp area can be filled with tertiary dentin, and since it induces the formation of tertiary dentin, MTA is considered a physiologically active substance. However, it has been found that MTA induces the formation of reparative dentin similar to bone, unlike physiological dentin or reactive dentin which has dentinal tubules.

[0129] Referring to Figure 6, it can be confirmed that at both day 7 and day 14, the group treated with both MTA and KH001 showed a statistically significant increase in absorbance compared to the control group. On day 14, the calcification ability of the group treated with both MTA and KH001 was statistically significantly different from that of the group treated with MTA alone.

[0130] (Examples 3-5. Evaluation of wound healing ability and cell migration ability after treatment with dental pulp capping composition) Human medullary cells (passage 2-4) are placed in a 6-well plate in a 1 × 10⁶ layer. 5 After inoculation at a density of cells / well, the cells were cultured in 10% α-MEM.

[0131] The day after cell inoculation, the central part of each well was scraped off using a 200 μL pipette tip to create a cell-free "wound area."

[0132] 24 hours later, images were taken using a 10x objective lens on an upright microscope, and each image was analyzed using the ImageJ program. All experiments were performed in three sets.

[0133] Figure 7 shows the results of examining changes in wound healing ability and cell migration ability in human dental pulp cell experiments treated with MTA-modified medium, in order to confirm the efficacy of KH001.

[0134] Pulp capping, which involves the application of MTA, is performed when there is damage to odontoblasts and pulp cells. This condition represents a kind of "wound" in the pulp, and requires a process of cell loss or the accumulation of surrounding cells at the damaged site. Therefore, as a cell experiment model that mimics this process, we investigated the changes in wound healing ability and cell migration ability caused by pulp capping compositions using a wound healing model.

[0135] Referring to Figure 7, wound healing analysis was performed to investigate the effects of KH001 on the wound healing ability and cell migration ability of odontoblasts in an environment treated with MTA. Although not statistically significant, the group treated with MTA tended to have inferior cell migration ability compared to the control group (Ctrl). In contrast, the group treated with both MTA and KH001 showed a tendency for cell migration ability to recover to a level similar to that of the control group. These results suggest that in situations where the dental pulp is exposed, treatment with a mixture of MTA and KH001 is more advantageous than applying MTA alone.

[0136] (Examples 3-6. Evaluation of dentinal tubule permeability of pulp capping composition (MTA prototype)) A. Preparing for a Beagle For the in vivo study, a total of 10 two-year-old beagle dogs were used. Depending on the periodontal condition of each beagle dog, 4 to 6 maxillary premolars and 6 mandibular premolars were used.

[0137] The control and experimental groups were assigned to separate beagle dogs to eliminate potential errors due to individual nutrition, behavior, and dental pulp conditions. All animal experiments were conducted according to protocols approved by the Animal Care Committee of Seoul National University Veterinary Hospital (SNU-180416-2-1, SNU-171020-5-2).

[0138] I. Evaluation of cell permeability After mixing a liquid containing 10 μg / mL of CPNE7 functional peptide (KH001) in vivo with ENDOCEM MTA powder according to the manufacturer's instructions, the mixture was applied to the cervical cavity near the pulp of a Beagle canine tooth, and the dentinal tubule permeability of the pulp capping composition (MTA prototype) was examined.

[0139] A dish-shaped V-class cavity was formed in the cervical region of a Beagle dog's dentition to a depth of approximately half the diameter of a #4 round bur. Then, MTA containing CPNE7 functional peptide (KH001) linked to fluorescent rhodamine was applied and fixed with 4% PFA. A test specimen was then prepared using a digital low-speed diamond cutting machine to a thickness of 0.1 mm in the sagittal plane.

[0140] Figure 8 shows photographs taken with a confocal microscope to examine the dentinal tubule permeability of the pulp capping composition. Figure 8A is a schematic diagram of the specimen preparation process, and B and C show the results of examining whether KH001 reached the dentin and pulp, respectively.

[0141] Referring to Figure 8, it can be seen that when 10 ug / mL of CPNE7 functional peptide was mixed directly with ENDOCEM MTA powder, the CPNE7 functional peptide flowed well along the dentinal tubules and successfully reached the dental pulp.

[0142] In actual cases where MTA is applied, even when a considerably thicker layer of dentin remains than when the pulp is exposed or only a thin layer of dentin remains close to the pulp, the CPNE7 functional peptide successfully reached the pulp through the dentinal tubules. Therefore, it can be expected that when performing pulp capping, a pulp capping composition to which the CPNE7 functional peptide is applied will seal the exposed pulp and provide an effective restorative effect.

[0143] This research was conducted under the 2021 National Research and Development Project of the Ministry of Science and ICT, the Ministry of Health and Welfare and the Ministry of Trade and Industry (KEIT) of the Republic of Korea (171113808080, 1711138079; "Development of Next-Generation Dental Restorative and Adhesive Materials that Induce Tooth Dentin Regeneration").

[0144] This specification and drawings disclose preferred embodiments of the present invention, and although specific terms are used, these are merely general terms used to illustrate the technical content of the invention and to aid in understanding the invention, and are not intended to limit the scope of the invention. It will be obvious to those ordinary skill in the art to which the present invention pertains that other modifications based on the technical idea of ​​the present invention are also possible, in addition to the embodiments disclosed herein.

Claims

1. A dentin bonding composition comprising a peptide consisting of the amino acid sequence of the following general formula 1: KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the above general formula 1, R1 is arginine (R), lysine (K), or glutamine (Q). R2 is either arginine (R) or glutamine (Q), R3, R4, and R5 are arginine (R) or lysine (K), respectively. R6 is either asparagine (N) or serine (S). R7 and R8 are lysine (K) or tyrosine (Y).

2. The dentin bonding composition is one of the amino acid sequences of SEQ ID NOs: 1 to 96. A dentin bonding composition according to claim 1, characterized by the following:

3. The dentin bonding composition according to claim 1, characterized in that the composition comprises a polypeptide in which the peptide is repeatedly linked.

4. The dentin bonding composition according to claim 1, wherein the composition is used to bond dental restorative materials to damaged tooth structure.

5. The dentin bonding composition according to claim 4, characterized in that the damaged tooth structure is carious or fractured tooth structure.

6. The dentin bonding composition according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

7. A method for preventing post-treatment tooth necrosis, tooth removal, or tooth loss by administering the composition described in claim 1 to an individual other than a human to improve or treat damaged tooth structure.

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