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

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 that contain a peptide derived from CPNE7, which forms biocompatible tubular dentin and tubular dentin having a continuous structure. [Background technology]

[0002] Dental restorative materials differ from teeth in various physical properties (thermal expansion coefficient, strength, wear resistance, elastic modulus, etc.), and processes such as tooth removal and chemical tooth treatment are required to physically and chemically match the materials to the tooth. Chemical irritation, microleakage, and irritation during treatment from dental materials for conservation and prosthetic restorations can weaken or cause existing pathological dental problems (fractures, hypersensitivity, caries, etc.), including sensitivity and pulp lesions. This can lead to necrosis of the living tissue within the tooth, resulting in root canal treatment to remove the pulp and subsequent tooth removal and loss. Furthermore, tooth sensitivity can occur when dentin is damaged for various reasons, exposing the dentinal tubules. This symptom can also occur when tooth tissue is removed during dental treatment.

[0003] To reduce hypersensitivity symptoms caused by exposed dentinal tubules, dentinal tubule closure and physiological mineralization are necessary. To overcome this limitation, the concept of physiological dentin sealing has been introduced, and new restorative materials with physiological activity that promote the recovery of odontoblasts, which are responsible for dentin secretion, are needed.

[0004] Geriatric dental diseases associated with aging have recently emerged as a serious social issue, and the expanded application of national health insurance to the elderly population is also affecting the dental field. Various research results have shown that the number of natural teeth in the elderly tends to decrease with age, and the fewer natural teeth there are, the lower the quality of life. Therefore, there is a growing demand for procedures and materials that can preserve patients' natural teeth as much as possible.

[0005] Teeth are composed of hard tissues such as enamel, dentin, and chalk, and the internal soft tissue known as the dental pulp. The dental pulp is home to well-developed nerves and blood vessels, and odontoblasts, which form dentin, are located at the boundary between the pulp and dentin. When the dental pulp is damaged for various reasons, such as caries or fractures, the symptoms experienced by the patient and the required treatment procedures vary depending on the extent of the damage. When damage is limited to the enamel or dentin, the weakened tissue is removed and then replaced with a restorative material. When the damage is close to the dental pulp or has invaded the pulp, and only a small area of ​​exposed pulp is exposed, the dental pulp can be directly sealed using a pulp capping material and repaired.

[0006] Restoration and prosthetic restorative materials made of various components, such as amalgam, glass ionomer cement, composite resin, gold, and porcelain, are used to restore tooth structure and function after tooth damage. Resin is currently the most commonly used material for direct restorations, and a dentin adhesive is used to attach the resin to the tooth. Eighth-generation adhesives, which shorten the application process, are currently the most common, and a representative eighth-generation adhesive is Bisco Dental's All-Bond Universal product. The development of adhesives with biological effects that restore damaged dentin thickness, prevent microleakage, and induce the restoration of damaged dentin thickness during resin restorations could help protect natural teeth in the future, thereby improving overall oral health and quality of life.

[0007] When a small area of ​​exposed pulp is present or the remaining tooth structure is thin and close to the pulp, pulp capping is performed to preserve pulp viability. An ideal pulp capping material would bond with the tooth structure without causing pulp inflammation, form a dentin bridge without microleakage, and be clinically easy to use. Many materials, including calcium hydroxide, mineral trioxide aggregate (MTA), and adhesive resins, have been investigated for successful pulp capping. Among these, MTA is most widely used due to its biocompatibility. One of the important functions of direct pulp capping materials is their biological effect, which stimulates odontoblasts within the pulp to induce new dentin formation. This effect allows for the sealing of the exposed pulp and prevents progression to nerve treatment. Nerve treatment requires the complete removal of the pulp within the tooth, ultimately making it difficult to preserve the natural tooth. Therefore, the development of a direct pulp capping material with dentin formation-inducing properties is necessary for the promotion of overall oral health.

[0008] Resin restoratives are commonly used, and although they have good adhesion to tooth structure, the toxicity of the monomers often causes patients to experience discomfort after restoration. To minimize side effects associated with treatment, it is necessary to seal exposed dentinal tubules, restore damaged dentin thickness, and minimize the effects of the monomer on the dental pulp. MTA, which is widely used as a pulp capping material, also has good biocompatibility and sealing power, but forms reparative dentin that differs from the existing tubular dentin. Over the long term, this reparative dentin can develop microleakage at the boundary with the existing dentin. Summary of the Invention [Problem to be solved by the invention]

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

[0010] Another object of the present invention is to provide a pulp-capping composition that can be directly applied to seal and repair the dental pulp when the damaged area of ​​a tooth is close to the dental pulp or when the dental pulp is invaded and the area of ​​exposed pulp is small.

[0011] The objects of the present invention are not limited to those mentioned above, and other objects not mentioned will be clearly understood by those having 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 above technical problems, there is provided a dentin bonding composition comprising 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 the 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), respectively; R6 is asparagine (N) or serine (S); R7 and R8 are lysine (K) or tyrosine (Y).

[0013] With regard to dentin adhesion, variant peptides having a sequence that differs by one or more amino acid residues from the amino acid sequence that constitutes them are also included in the category of peptides provided by the present invention, as long as they can exhibit the effect of promoting physiological mineralization of dentin.

[0014] In general, amino acid exchanges in proteins and polypeptides that do not change the overall activity of the molecule are known in the art. 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. Furthermore, peptides may be included that have been modified or mutated in the amino acid sequence to increase the structural stability of the peptide against heat, pH, etc., or to increase the ability to promote dentin synthesis.

[0015] For example, the acidic amino acid glutamine at position 3 of the peptide of SEQ ID NO: 1 can be substituted with the basic amino acid lysine or arginine, and the peptide of the present invention still exhibits the same effects; the basic amino acid arginine at position 4 or 5 of the peptide of SEQ ID NO: 1 can be substituted with the acidic amino acid glutamine or the basic amino acid lysine, and the peptide of the present invention still exhibits the same effects; the basic amino acid lysine at position 6, 7, or 9 of the peptide of SEQ ID NO: 1 can be substituted with the basic amino acid arginine or the aromatic amino acid tyrosine, and the peptide of the present invention still exhibits the same effects; the acidic amino acid asparagine at position 8 of the peptide of SEQ ID NO: 1 can be substituted with the neutral amino acid serine, and the peptide of the present invention still exhibits the same effects; and the aromatic amino acid tyrosine at position 10 of the peptide of SEQ ID NO: 1 can be substituted with the basic amino acid lysine, and the peptide of the present invention still exhibits the same effects.

[0016] Thus, even if the acidic amino acids, basic amino acids, or aromatic amino acids constituting the peptides of the present invention are substituted with different acidic amino acids, basic amino acids, neutral amino acids, or aromatic amino acids, the effects of the peptides provided by the present invention can be exhibited as they are. Therefore, it is obvious that mutant peptides having a sequence that differs by one or more amino acid residues from the amino acid sequence constituting the peptides of the present invention are also included in the category of peptides provided by the present invention.

[0017] Furthermore, the peptides of the present invention can exhibit the same effects as the peptides provided by the present invention even if they have any amino acids added to their N- or C-terminus. For example, the peptide may have 1 to 300 amino acids added to its N- or C-terminus, or 1 to 100 amino acids added to its N- or C-terminus, or 1 to 24 amino acids added to its N- or C-terminus.

[0018] Specifically, the peptides according to general formula 1 can be represented in Tables 1 to 12 below.

[0019] For example, a 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 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 peptide of SEQ ID NO: 1 or amino acids 5 to 7 of the peptide of SEQ ID NO: 1 with lysine or arginine (Table 1).

[0021] [Table 1]

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

[0023] [Table 2]

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

[0025] [Table 3]

[0026] Group 4 peptides can then be synthesized by substituting amino acids 5-7 of the peptide of SEQ ID NO:1 with lysine or arginine, the eighth amino acid with serine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 4).

[0027] [Table 4]

[0028] Next, the peptides of Group 5 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, and the fifth through seventh 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 through 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 through 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 through 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 through 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 through 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 through 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 may 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 through 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 aspect, the present invention provides a polynucleotide encoding the peptide.

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

[0046] In another aspect, 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] The term "expression vector" as used herein refers to a recombinant vector capable of expressing a target peptide in a target host cell, comprising essential regulatory elements operably linked to a gene insert for expression. The expression vector includes expression regulatory elements such as an initiation codon, a termination codon, a promoter, and an operator. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the polypeptide, and must be functional in an individual when the gene product is administered, and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.

[0048] As used herein, the term "operably linked" refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked so as to perform their common function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect the expression of the coding sequence. Operable linkage with an expression vector can be produced using recombinant DNA techniques known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes commonly known in the art.

[0049] Additionally, the expression vector may contain a signal sequence for peptide excretion to facilitate separation of the peptide from the cell culture medium. Specific initiation signals may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, exogenous translational control signals, which may include the ATG initiation codon, must be provided. These exogenous translational control signals and initiation codons may be of various natural and synthetic origins. Expression efficiency may be increased by the introduction of appropriate transcriptional or translational enhancing factors.

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

[0051] The term "tag" as used herein refers to a molecule that exhibits a quantifiable activity or property, and may be a fluorescent molecule, including a chemical fluorescein, a polypeptide fluorescein, such as green fluorescent protein (GFP) or related proteins, or an epitope tag, such as a Myc tag, Flag tag, histidine tag, leucine tag, IgG tag, or streptavidin tag. In particular, when an epitope tag is used, a peptide tag consisting of preferably 6 or more amino acid residues, more preferably 8 to 50 amino acid residues, may be used.

[0052] In the present invention, the expression vector may contain a nucleotide sequence encoding the peptide of the present invention that provides the effect of promoting physiological mineralization of dentin. The vector used in this case is not particularly limited as long as it is capable of producing the peptide. Preferably, the vector is a plasmid DNA, a phage DNA, or the like. More preferably, commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retrovirus, adenovirus, vaccinia virus, etc.), and the like. The expression vectors may be vectors of various types, such as insect virus, insect virus vectors (e.g., baculovirus), etc. Since the expression level and modification of proteins vary depending on the host cell, it is desirable to select and use the host cell that is most suitable for the purpose.

[0053] The transformant provided by the present invention can be prepared by transforming a host with the expression vector provided by the present invention, and can be used to produce the peptide by expressing the polynucleotide contained in the expression vector. The transformation can be carried out by various methods, including, but not limited to, CaCl precipitation, the Hanahan method in which the efficiency of CaCl precipitation is improved by using a reducing agent such as DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and desiccation / repression-mediated transformation. Furthermore, the host used to prepare the transformant is not particularly limited as long as it is capable of producing the peptide, and may be bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc.; yeast cells such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, etc.; fungal cells such as Pichia pastoris, insect cells such as Drosophila and Spodoptera frugiperda Sf9 cells, etc.; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells, or plant cells.

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

[0055] The term "culture" as used herein refers to a method of growing microorganisms under artificially controlled environmental conditions. In the present invention, the method of culturing the transformant can be performed by a method known in the art. Specifically, the culture is not particularly limited as long as it can express and produce the peptide of the present invention that promotes physiological mineralization of dentin, and may be continuously cultured by batch, fed-batch, or repeated fed-batch process.

[0056] The medium used for cultivation must meet the requirements of the specific strain by adjusting the temperature, pH, etc. under aerobic conditions in a standard medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. Carbon sources that can be used include a mixed sugar 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 alone or in combination. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, as well as amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its hydrolyzed products, and defatted soybean cake or its hydrolyzed products. These nitrogen sources can be used alone or in combination. The medium can contain potassium monophosphate, potassium diphosphate, and the corresponding sodium-containing salts as phosphorus sources. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium-containing salts, can be used as phosphorus sources. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the above substances.

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

[0058] Additionally, foam formation can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. To maintain an aerobic state, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture. The temperature of the culture is usually 27°C to 37°C, preferably 30°C to 35°C. The culture is continued until the maximum amount of the peptide is produced. To achieve this goal, the culture is usually continued for 10 to 100 hours.

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

[0060] The term "prevention" as used herein means any action of inhibiting or delaying the occurrence of pulp necrosis, tooth removal or tooth loss, or secondary caries or pulpitis that may occur due to dental restorative materials, by administering a dentin bonding composition or pulp capping composition containing the peptide of the present invention.

[0061] The term "treatment" as used herein means any action of administering a restorative material, a dentin bonding composition, or a pulp capping composition, including a dentin bonding composition containing the peptide of the present invention as an active ingredient, to an individual seeking dental restorative treatment, thereby promoting physiological remineralization of dentin between the tooth and the restorative material, thereby enabling dental restorative treatment to be performed.

[0062] The composition of the present invention may be prepared in the form of a composition for treating dental tissue damaged by caries, fractures, or the like, further comprising a suitable carrier (natural or non-natural carrier), excipient, or diluent commonly used in preparing dental restoration compositions. Specifically, the composition may be formulated into a sterile injectable solution that can be administered to the site of dental tissue damage, such as caries or fractures, by a conventional method. Examples of carriers, excipients, and diluents that may be included in the composition 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, collagen, etc. When formulated, they may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. In particular, these may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, ointments (e.g., dental pulp liner, etc.), etc. Examples of non-aqueous solvents and suspensions that may be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of suppository bases that may be used include witepsol (registered trademark), macrogol (registered trademark), Tween (registered trademark) 61, cocoa butter, lauric butter, and glycerogelatin.

[0063] The content of the peptide contained 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 a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level can be determined based on factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, and sex, the patient's sensitivity to the drug, the administration time, route of administration, and excretion rate of the composition of the present invention used, the duration of treatment, and drugs used in combination with or concomitantly with the composition of the present invention, as well as 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 teeth, such as caries and fractures. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum efficacy at the minimum dose without adverse 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 purpose of use, severity of the disease, the patient's age, weight, sex, medical history, and the type of substance used as the active ingredient. For example, the composition of the present invention can be administered at a dose of about 0.1 ng / kg to about 100 mg / kg, preferably 1 ng / kg to about 10 mg / kg, per adult. The administration frequency of the composition of the present invention is not particularly limited, but it can be administered once a day or in divided doses administered several times a day. The above 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 tissue such as caries or fractures, comprising the step of administering a pharmaceutically effective amount of the composition to an individual suffering from damaged tooth tissue such as caries or fractures.

[0067] The term "individual" as used herein may include, without limitation, mammals including rats and livestock that require treatment for damaged teeth such as caries and fractures, but humans may be excluded from the individuals that have developed the disease.

[0068] The administration route of the composition for treating damaged tooth tissue, such as caries or fractures, of the present invention can be any common route as long as it can reach the target tissue. The composition of the present invention can be provided in any dosage form suitable for topical application depending on the purpose, including, but not limited to, oral, transdermal, intravenous, intramuscular, or subcutaneous administration. The composition can be in the form of an injection, a solution for topical application, a suspension, an emulsion, a gel, a puff, or a spray, but is not limited to these. The dosage form can be easily prepared according to conventional methods in the art, and surfactants, excipients, hydrating agents, emulsifiers, suspending agents, salts or buffers for adjusting osmotic pressure, coloring agents, spices, stabilizers, antiseptics, preservatives, or other commonly used adjuvants can be used as appropriate.

[0069] In another aspect, the present invention provides a quasi-drug composition for preventing or improving tooth structure damaged by caries, fractures, etc., comprising the peptide.

[0070] The term "amelioration" according to the present invention refers to any action that at least reduces a parameter related to the condition being treated, such as the severity of the symptoms.

[0071] In the present invention, the improvement can be interpreted as meaning any action of administering a composition containing the peptide of the present invention as an active ingredient to an individual who requires treatment for damaged teeth such as caries or fractures, thereby promoting the synthesis of dentin and thereby improving or benefiting the symptoms of damaged teeth such as caries or fractures.

[0072] The term "quasi-drug" as used herein means an article used for the purpose of diagnosing, curing, improving, mitigating, treating, or preventing a disease in humans or animals, which has a milder effect than a pharmaceutical product. For example, according to the Pharmaceutical Affairs Law, quasi-drugs exclude articles used for pharmaceutical purposes, and include fiber and rubber products used to treat or prevent diseases in humans and animals, items that have a mild effect on the human body or do not have a direct effect on the human body and are not tools or machines, or items similar to them, and disinfectants and insecticides used to prevent infectious diseases.

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

[0074] In another aspect, the present invention provides a health functional food composition for preventing or improving damaged tooth structure such as caries or fractures, which contains the peptide.

[0075] The term "food" as used herein includes all foods in the usual sense, such as meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, health supplements, alcoholic beverages, vitamin complexes, functional health foods, and health foods.

[0076] The term "functional food" refers to a food with high medical and therapeutic value that is processed to efficiently exert bioregulatory functions in addition to providing nutrients. The term "functional" refers to the regulation of nutrients or the physiological effects on the structure and function of the human body. The food of the present invention can be prepared by methods commonly used in the art, and may be prepared by adding raw materials and ingredients commonly used in the art. The dosage form of the food may also be any dosage form recognized as a food. The food composition of the present invention can be prepared in various dosage forms. Unlike general pharmaceuticals, the food composition is made from food ingredients, which has the advantage of being free of 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 adjuvant to promote the prevention or improvement of damaged teeth, such as caries and fractures.

[0077] The term "health food" refers to food that has more active health maintenance and promotion effects than general foods, and "health supplement food" refers to food for the purpose of health supplementation. In some cases, the terms "health functional food," "health food," and "health supplement" may be used interchangeably.

[0078] Specifically, the health functional foods are foods in which the peptides of the present invention are added to food ingredients such as beverages, teas, spices, gums, and confectioneries, or are manufactured as capsules, powders, suspensions, etc., and when ingested, they bring about specific health benefits. However, unlike general medicines, they have the advantage of being made from food ingredients and therefore free from side effects that can occur when taking medicines for a long period of time.

[0079] The food composition of the present invention can be taken on a daily basis, and is therefore expected to be highly effective in preventing or improving damaged tooth structures such as caries and fractures, making it highly useful.

[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] The food composition may also contain additional ingredients commonly used in food compositions to improve smell, taste, visual appearance, etc. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, etc. Minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr) may also be included. Amino acids such as lysine, tryptophan, cysteine, and valine may also be included.

[0082] The food compositions may further contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high-strength bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color formers (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 D-bitartrate, etc.), fortifiers, emulsifiers, thickeners (thickening agents), coating agents, gum bases, antifoaming agents, solvents, and improvers. The additives may be selected according to the type of food and used in appropriate amounts.

[0083] The peptides of the present invention can be added directly or in combination with other foods or food ingredients, and can be used appropriately in a conventional manner. The amount of active ingredient to be added can be determined appropriately depending on the intended use (prevention, health, or therapeutic treatment). Generally, when producing a food or beverage, 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, when taking the food or beverage for long-term health and hygiene purposes, the active ingredient can be added in an amount below the above range, and there is no problem from the standpoint of safety, so the active ingredient can be used in an amount above the above range.

[0084] One example of the food composition of the present invention may be a health drink composition. In this case, similar to conventional beverages, various flavorings or natural carbohydrates may be added as additional ingredients. The 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. Examples of sweeteners that may be used include natural sweeteners such as thaumatin and stevia extract; and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates per 100 mL of the health drink composition of the present invention may typically be about 0.01 g to 0.04 g, specifically about 0.02 g to 0.03 g.

[0085] In addition to the above, the health drink composition may contain various nutrients, vitamins, electrolytes, flavorings, coloring agents, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. Fruit pulp may also be included for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not critical, but is typically selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health drink composition of the present invention.

[0086] The food composition of the present invention may contain the peptide of the present invention in various weight percentages as long as it can prevent or improve tooth damage such as caries and fractures. Specifically, the food composition may contain the peptide of the present invention in an amount of 0.00001 to 100 weight % or 0.01 to 80 weight % based on the total weight of the food composition, but is not limited thereto. [Effects of the Invention]

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

[0088] When the area of ​​tooth damage is close to the dental pulp or the dental pulp has been invaded, and the area of ​​exposed pulp is small, the dental pulp capping composition according to the embodiment of the present invention can be directly applied to seal the dental pulp and repair it, thereby enabling the sealing of the dental pulp and preventing or suppressing microleakage that may occur at the boundary of the repair dentin, thereby providing highly reliable and sustainable treatment of dental damage.

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

[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. Marker genes included DSPP, DMP1, and BSP, and gene expression was measured by RT-PCR. All results are shown as the mean and standard deviation of triplicate experimental results, with *p<0.05 compared to the control group. [Figure 2] Figure 2 shows the results of measuring the dentinal tubule permeability to determine whether KH001 successfully reaches the dental pulp when a mixed solution of dentin adhesive and KH001 is applied to a human tooth with exposed dentin. A is a schematic diagram of the sample preparation process for measuring dentinal tubule permeability, and B to D are images taken with a confocal microscope. [Figure 3]Figure 3 shows the results of a histological evaluation of the tubular dentin regeneration ability of a mixed solution of dentin adhesive and KH001. After creating a dish-shaped injury in the cervical region of Beagle teeth to expose the dentin, the control group was treated with a dentin adhesive that did not contain KH001 and then restored with resin, while the experimental group was treated with a dentin adhesive that contained KH001 and then restored with resin. Histological evaluation was then performed using H&E (hematoxylin eosin) staining to examine the formation of tubular dentin. The dotted line indicates the boundary of the newly formed tubular dentin, and TD (tertiary dentin) in the figure refers to tubular dentin. [Figure 4] Figure 4 shows the results of examining the expression levels of odontogenic differentiation marker genes in human dental pulp cells at various concentrations of KH001 under MTA conditioned medium conditions. Marker genes included DSPP, DMP1, and BSP, and gene expression was measured by RT-PCR. All results are shown as the mean and standard deviation of triplicate experimental results, with *p<0.05 and **p<0.01 indicating results compared to the control group. [Figure 5a] Figure 5a shows the results of examining the expression changes of odontoblast differentiation gene markers on day 7 of differentiation to confirm the efficacy of KH001 in an experiment using human dental pulp cells treated with MTA-conditioned medium. [Figure 5b] Figure 5b shows the results of examining the expression changes of odontoblast differentiation gene markers on day 14 of differentiation to confirm the efficacy of KH001 in an experiment using human dental pulp cells treated with MTA-conditioned medium. [Figure 6] Figure 6 shows the results of examining changes in odontoblast differentiation and mineralization ability in an experiment using human dental pulp cells treated with MTA-conditioned medium to confirm the efficacy of KH001. [Figure 7] FIG. 7 shows the results of examining changes in wound healing ability and cell migration ability in an experiment using human dental pulp cells treated with MTA-conditioned medium to confirm the efficacy of KH001. [Figure 8]FIG. 8 is a photograph taken with a confocal microscope to examine the ability of the pulp-capping composition according to an embodiment of the present invention to penetrate dentinal tubules in dentin. [Figure 9] FIG. 9 is a schematic diagram showing the sites where the dentin bonding composition and the pulp capping composition are applied in the process of treating a damaged tooth, and the appearance before and after application. DETAILED DESCRIPTION OF THE INVENTION

[0091] The objects and advantages of the present invention, as well as the technical configurations for achieving them, will become apparent from the following detailed description of the embodiments together with the accompanying drawings. In describing the present invention, if it is determined that a detailed description of a known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted. The terms used below are defined for describing the embodiments of the present invention, and may vary depending on the intentions or practices of users and operators.

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

[0093] Examples of the present invention will be specifically described below. Example 1: Materials and Methods [Example 1-1: Preparation of peptide] The KH001 peptide (SEQ ID NO: 96) is a synthetic peptide corresponding to a 10-amino acid fragment (KYKQKRRSYK) of the hCPNE7 protein, spanning residues 344 to 353. The peptide KH001 was synthesized using an Fmoc (9-fluorenylmethoxycarbonyl)-based solid-phase method, and the purity of the peptide KH001 was determined to be 97% or higher by high-performance liquid chromatography.

[0094] [Example 1-2: Cell culture] MDPC-23 (mouse pre-odontoblast cell line) cells were provided by Dr. JE Nor (University of Michigan, Ann Arbor, MI, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Carlsbad, CA, USA). C3H10T1 / 2 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI 1640 medium (Gibco BRL). Both cell lines were cultured at 37°C in a 5% CO2 atmosphere, supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL) and antibiotic-antimycotic supplements (Gibco BRL).

[0095] Cells were cultured in minimum essential medium alpha (MEM-α; Gibco BRL) for in vitro and ex vivo experiments. For differentiation of human dental pulp cells (hDPCs) and MDPC-23 cells, 80%–90% fused cells were cultured for 3 weeks in the corresponding 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 dentin bonding composition] KH001 and Bisco Dental's (registered trademark) dentin adhesive ABU (All-Bond Universal) were mixed together, with 2 mg of KH001 added per mL of ABU, and the mixture was stirred for approximately 40 minutes under reduced pressure (-750 mmHg) using a stirrer (stirring conditions: Paddle 10-30 rpm, Disperse 500-600 rpm, Homo 2400-3200 rpm). The mixture was then stored in a light-shielding container. The prepared dentin bonding composition was refrigerated at 4°C for approximately 3 months after preparation and was applied to Examples 2-3 and 2-4, and is shown in Table 13.

[0097] [Example 2-1-2: Preparation of dentin bonding composition] In the cell experiments on human dental pulp cells (hDPCs) prepared in Example 1-2, ABU was diluted 1:5000 with 10% α-MEM to prevent side effects such as cell death due to the toxicity of the dentin adhesive monomer. KH001 peptide powder was dissolved in the diluted solution to prepare concentrations of 5 μg / ml and 10 μg / ml, respectively, and the dentin adhesive compositions prepared in this manner were used in 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] After treating the human dental pulp cells (hDPCs) prepared in Example 1-2 with the dentin bonding composition of Example 2-1-2, the expression of the 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 shown as the average and standard deviation of the results of three experiments, with *p<0.05 and **p<0.01 indicating results compared to the control group.

[0099] As shown in Figure 1, the expression levels of odontoblast differentiation marker genes DSPP, BSP, and DMP1 increased as the concentration of KH001 increased, and when treated with KH001 at a concentration of 10 μg / 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 dentinal tubule permeability was observed. The extracted human teeth were impacted human third molars from patients aged 18 to 22 years old, provided by Seoul National University Dental Hospital. The experimental protocol was approved by the Institutional Review Board (IRB number: S-D20140007), and prior informed consent was obtained from all patients.

[0101] The crown of an extracted human tooth was cut transversely with a diamond saw to expose the dentinal tubules, and then the dentin bonding composition prepared in Example 2-1-1 containing KH001 peptide (sequence number 96) with rhodamine as a fluorescent staining reagent was applied to the coronal surface of the extracted human tooth.

[0102] FIG. 2 shows the results of a confocal image taken after applying the dentin bonding composition without brushing or air drying, leaving it for about 30 seconds, and then photo-polymerizing it.

[0103] Figure 2 shows the results of measuring the dentinal tubule permeability to determine whether KH001 successfully reaches the dental pulp when a mixed solution of dentin adhesive and KH001 is applied to a human tooth with exposed dentin for approximately 30 seconds. Referring to Figure 2, A is a schematic diagram of the sample preparation process for measuring dentinal tubule permeability. Images 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 dental pulp cavity.

[0104] [Example 2-4: Histological evaluation of tubule dentin regeneration ability] After creating a dish-shaped injury to the cervical region of the teeth of beagle dogs to expose the dentin, the control group was treated with a dentin adhesive that was not mixed with the peptide KH001, followed by a resin restoration. The experimental group was treated with a dentin adhesive containing KH001, followed by a resin restoration. Histological evaluation was then performed using H&E staining to examine the formation of tubular dentin.

[0105] Figure 3 shows the results of histological evaluation of the tubular dentin regeneration ability of the dentin bonding composition of Example 2-1-1. Referring to Figure 3, the dotted line indicates the boundary of newly formed tubular dentin, and TD (newly formed tertiary dentin) is dentin with newly formed dentinal tubules.

[0106] 3, in the control group (I), to which only the dentin adhesive (ABU) was applied, no dentin tubules were formed below the damaged dentin site, whereas in the test groups (II) (containing 5 μg / ml of KH001) and (III) (containing 10 μg / ml of KH001), to which the dentin bonding composition containing KH001 prepared in Example 2-1-2 was applied, dentin tubules were formed on the pulp side below the damaged dentin site. Furthermore, odontoblasts were lined up in a circle in the pulp adjacent to the newly formed dentin tubules, indicating an activated and healthy pulp.

[0107] Table 13 shows the results of a shear bond strength test using the Ultradent method to confirm the adhesive strength of the dentin bonding composition containing KH001 prepared in Example 2-1-1 to tooth tissue. After the dentin surface was smoothed with 320-grit sandpaper and 600-grit sandpaper, Bisco ABU (control group) and a dentin adhesive containing KH001 at a concentration of 2 mg / mL (experimental group) were applied using the self-etch method for approximately 10-15 seconds, and the adhesive was then applied at 1000 mW / cm. 2After photopolymerization with a light source of 1000 kJ / s, the specimens were scrubbed and then irradiated with a DLU (Duo-Link Universal®, Bisco) for 20 seconds with a light source of the same intensity to bond them. The specimens were then stored in DI water at 37°C before measurements were taken. Each group contained 10 specimens.

[0108] [Table 13]

[0109] Table 13 summarizes the adhesive strength of the dentin bonding composition prepared in Example 2-1-1. Referring to Table 13, even though KH001 was contained at a high concentration of about 200 times the effective concentration, the adhesive strength of the dentin bonding composition provided a similar level of adhesive strength to conventional dentin adhesives. These results suggest that even if KH001 is contained in a dentin bonding composition at a concentration of about 2 mg / mL or less, it can provide an adhesive strength at a level equivalent to that of conventional dentin adhesives.

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

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

[0112] Thereafter, the conditioned medium diluted at a ratio of 1 / 5 was treated with KH001 at concentrations of 5 μg / mL and 10 μg / mL, respectively, to prepare compositions for dental pulp capping.

[0113] Example 3-2: Evaluation of expression levels of odontogenic differentiation marker genes (DSPP, DMP1, BSP) and CPNE7 gene at different concentrations of KH001, a dental pulp capping composition Total RNA was extracted from human dental pulp cells using Tri-Reagent®, and then treated with reverse transcriptase and oligo(dT) primer (Invitrogen) to synthesize cDNA.

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

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

[0116] Figure 4 shows the results of RT-PCR analysis of the expression levels of odontoblast differentiation marker genes and CPNE7 gene in human dental pulp cells at various concentrations of the dental pulp capping composition KH001 (KH001 in MTA-conditioned medium). All values ​​are shown as the mean and standard deviation of triplicate experimental results, with *p<0.05 and **p<0.01 representing results compared to the control group.

[0117] Example 3-3: Evaluation of the differential expression levels of odontoblast differentiation marker genes (DSPP, DMP1, BSP) and CPNE7 gene on a daily basis following treatment with a dental pulp capping composition (KH001 10 μg / mL under MTA-conditioned medium conditions) To compare and measure the ability of the dental pulp capping composition containing 10 μg / ml of KH001 prepared in Example 3-1 to induce differentiation of human dental pulp cells into odontoblasts, a control group treated with differentiation medium only (Ctrl), a group treated with MTA-conditioned differentiation medium (MTA), and a group treated with the dental pulp capping composition (MTA-conditioned / CPNE7 functional peptide, MTA+KHOO1) were compared.

[0118] The differentiation medium was basically prepared so that it contained α-MEM supplemented with 5% FBS, 1% antibiotic, 10 mM β-glycerophosphate, and 50 μg / mL ascorbic acid.

[0119] After treating human dental pulp cells with each 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 the expression of odontoblast differentiation gene markers over the course of differentiation in an experiment using human dental pulp cells treated with MTA-conditioned medium to confirm the efficacy of KH001 (MTA+KH001). MTA is a formulation used when the dental pulp is exposed, or when the pulp is not exposed but only a thin layer of dentin remains, indicating potential pulp damage. Therefore, the odontoblasts originally present in the exposed pulp area are likely to die. Therefore, in order to recover and form tertiary dentin in the exposed pulp area, it is important to induce dental pulp cells to differentiate into odontoblasts.

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

[0122] (Example 3-4: Evaluation of odontoblast differentiation and mineralization ability by treatment with MTA-conditioned medium) To examine the effect of CPNE7 functional peptide on the differentiation and mineralization of odontoblasts in the MTA-treated environment, ARS (Alizarin red staining) was performed.

[0123] Human dental pulp cells (passages 2–4) were cultured in a 6-well plate at 1 × 10 5 After seeding at a density of 100 cells / well, when the cells reached approximately 80% confluency, they were treated with the corresponding differentiation medium for the control and experimental groups for 7, 14, and 21 days, and the differentiation medium was changed every 2 days.

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

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

[0126] The absorbance was then measured at 405 nm with an ARS standard, and all experiments were performed in triplicate.

[0127] Figure 6 shows the results of examining changes in odontoblast differentiation and mineralization ability in an experiment using human dental pulp cells treated with MTA-conditioned medium to confirm the efficacy of KH001 (a composition for dental pulp capping).

[0128] MTA is used clinically when the dental pulp is exposed and the odontoblasts have died. To maintain the pulp in this situation, pulp cells must be induced to differentiate into odontoblasts, and the differentiated odontoblasts must be able to secrete mineralizing substances. This process allows the exposed pulp area to be filled with tertiary dentin, which induces its formation. MTA is therefore considered a bioactive substance. However, unlike physiological or reactive dentin with dentinal tubules, MTA induces the formation of reparative dentin, which resembles bone.

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

[0130] (Example 3-5. Evaluation of wound healing ability and cell migration ability by treatment with dental pulp capping composition) Human marrow cells (passages 2–4) were cultured in a 6-well plate at 1 × 10 5 After seeding at a density of 100 cells / well, the cells were cultured in 10% α-MEM.

[0131] The day after cell seeding, the center of each well was scraped using a 200 μL pipette tip to create a cell-free "wound."

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

[0133] FIG. 7 shows the results of examining changes in wound healing ability and cell migration ability in an experiment using human dental pulp cells treated with MTA-conditioned medium to confirm the efficacy of KH001.

[0134] Pulp capping, which involves the application of MTA, is performed when odontoblasts and pulp cells have been damaged. This condition is a kind of "wound" in the pulp, and must be preceded by a process in which cells are lost or surrounding cells gather at the damaged area. As a cellular experimental model that mimics this process, we investigated the changes in wound healing ability and cell migration ability caused by the pulp capping composition using a wound healing model.

[0135] Referring to Figure 7, a wound healing assay was performed to examine the effects of KH001 on the wound healing and cell migration ability of odontoblasts in an MTA-treated environment. The results showed that, although not statistically significant, the MTA-treated group tended to exhibit lower cell migration ability compared to the control group (Ctrl). In contrast, the group treated with MTA and KH001 together demonstrated a tendency for cell migration ability to recover to a similar level to the control group. These results suggest that in situations where dental pulp is exposed, applying a combination 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 the Beagle A total of 10 beagle dogs (2 years old) were used for the in vivo study. Depending on the periodontal status of each beagle dog, 4 to 6 maxillary premolars and 6 mandibular premolars were used.

[0137] Each beagle dog was assigned to a control group and an experimental group to eliminate potential errors due to individual nutrition, behavior, and dental pulp conditions. All animal experiments were conducted in accordance with protocols approved by the Animal Care and Use Committee of Seoul National University Animal Hospital (SNU-180416-2-1, SNU-171020-5-2).

[0138] B. Cell penetration ability evaluation A liquid containing 10 μg / mL of CPNE7 functional peptide (KH001) in vivo was mixed with ENDOCEM MTA powder according to the manufacturer's instructions, and then applied to the cervical cavity close to the dental pulp of a beagle canine tooth. The dentinal tubule penetration ability of the pulp-capping composition (MTA prototype) was then examined.

[0139] A dish-shaped V-class cavity was created in the cervical region of a beagle dog's tooth to a depth of about half the diameter of a #4 round bur. MTA containing the fluorescent rhodamine-linked CPNE7 functional peptide (KH001) was then applied and fixed in 4% PFA. Test specimens were then cut to a thickness of 0.1 mm in the sagittal plane using a digital low-speed diamond saw.

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

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

[0142] Although a significantly thicker layer of dentin remained than in the actual application of MTA, where the pulp is exposed or the dentin is so thin that it is close to the pulp, the CPNE7 functional peptide successfully reached the pulp through the dentinal tubules. Therefore, when performing pulp capping surgery, it is expected that the pulp capping composition containing the CPNE7 functional peptide will seal the exposed pulp and provide effective restorative effects.

[0143] This study was supported by the National Research and Development Project (171113808080, 1711138079; "Development of next-generation dental restorative and adhesive new materials that induce tooth dentin regeneration") of the Ministry of Science, ICT, Welfare, and Industry (KEIT) of the Republic of Korea in fiscal year 2021.

[0144] In the present specification and drawings, preferred embodiments of the present invention are disclosed, and although specific terms are used, these are used in a general sense merely to clearly explain the technical contents of the present invention and to aid in understanding the invention, and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art to which the present invention pertains that, in addition to the embodiments disclosed herein, other modifications based on the technical concept of the present invention can be implemented.

Claims

1. A dentin bonding composition comprising 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 the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4 and R5 are each arginine (R) or lysine (K); R6 is asparagine (N) or serine (S); R7 and R8 are lysine (K) or tyrosine (Y).

2. The dentin bonding composition according to claim 1, characterized in that the dentin bonding composition has an amino acid sequence of any one of SEQ ID NOs: 1 to 96.

3. A composition for dental pulp capping comprising MTA (Mineral Trioxide Aggregate) and 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 the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4 and R5 are each arginine (R) or lysine (K); R6 is asparagine (N) or serine (S); R7 and R8 are lysine (K) or tyrosine (Y).

4. The composition for dental pulp capping according to claim 3, characterized in that the composition for dental pulp capping has any one of the amino acid sequences of SEQ ID NOs: 1 to 96.

5. A polynucleotide encoding the peptide of claim 1 or 3.

6. An expression vector comprising the polynucleotide of claim 5.

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

8. The dentin bonding composition according to claim 1, wherein the composition is used for bonding a dental restorative material to damaged tooth tissue.

9. The dentin bonding composition according to claim 8, wherein the damaged tooth tissue is carious or fractured tooth tissue.

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

11. A method for preventing post-treatment necrosis of tooth tissue, tooth loss, or tooth loss by administering the composition of claim 1 or 3 to an individual other than a human to improve or treat damaged tooth tissue.

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