Composition for adhering dentin and composition for covering dental pulp, containing peptide derived from CPNE7

Abstract

A composition for adhering dentin according to the present invention comprises a peptide consisting of the amino acid sequence of the following general formula 1. K-Y-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 respectively 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 is used in the treatment of damaged teeth such as caries and fractures, thereby preventing dental pulp necrosis, tooth removal, or tooth loss that may occur after the treatment.

Description

Technical Field

[0001] The present invention relates to a dentin adhesive and a composition for covering a dental pulp, and more particularly, to a dentin adhesive containing a peptide derived from CPNE7 and a composition for covering a dental pulp, for forming tubular dentin having a continuous structure with biocompatible tubular dentin. Background Art

[0002] As for various dental restorative materials, they have various physical properties (thermal expansion coefficient, strength, abrasion resistance, elastic modulus, etc.) different from the teeth themselves. As a process to make the corresponding materials physically and chemically suitable for teeth, it is accompanied by processes such as tooth removal and chemical treatment of teeth. Chemical stimulation, slight leakage and stimulation during the treatment process of dental materials used for preservation and repair may cause or aggravate hyperesthesia and pulp lesions on existing pathological tooth problems (fractures, sensitivity, caries, etc.). This will cause necrosis of the pulp of the living tissue in the tooth, leading to root canal treatment to remove the pulp and further removal and loss of the tooth. In addition, when the dentin of the tooth is damaged due to various reasons and the dentinal tubules are exposed to the outside, the tooth will experience soreness. This symptom will also occur when tooth removal occurs during dental treatment.

[0003] In order to reduce the pain caused by exposed dentinal tubules, closure of dentinal tubules and physiological mineralization are required. In order to overcome these limitations, the concept of physiological dentine closure has emerged. Currently, new restorative materials with physiological activity are needed to promote the recovery of odontoblasts responsible for secreting dentin.

[0004] As aging has become a serious social issue recently, geriatric dental diseases have also had an impact on the dental field due to the expansion of national health insurance for the elderly population. Various research results have shown that the number of natural teeth in the elderly tends to decrease with age, and the fewer the number of natural teeth, the lower the quality of life. Therefore, there is an increasing demand for surgical procedures and materials that preserve the patient's natural teeth as much as possible.

[0005] A tooth is made up of enamel, dentin, and cementum, which are hard tissues, and pulp, which is the soft tissue inside. The nerves and blood vessels of the pulp are well developed, and the odontoblasts that form the dentin are located at the boundary between the pulp and the dentin. When the dentin is damaged due to various reasons such as caries and fractures, the symptoms felt by the patient and the required treatment methods will vary depending on the extent of the damage. If the damage to the tooth is limited to the enamel or dentin, the weakened dentin will be removed and then filled with restorative materials. When the range of damage is close to the pulp or invades the pulp, if the exposed pulp is small, the pulp will be sealed with direct pulp capping materials for repair.

[0006] In order to restore the structure and function of damaged teeth, preservation and repair materials composed of various components such as amalgam, glass ionomer cement, composite resin, gold, ceramics, etc. are used. Among them, the most commonly used material for direct restoration is resin, and dentin adhesive is used to attach the resin to the tooth. At present, the eighth-generation adhesive that reduces the coating steps is the most common, and the representative eighth-generation adhesive material is Bisco Dental's All-BondUniversal product. When restoring with resin, if an adhesive material can be developed that can restore the thickness of damaged dentin, prevent fine leakage, and have a biological effect of inducing the restoration of the thickness of damaged dentin, it will help protect natural teeth in the future, which can improve overall oral health and quality of life.

[0007] When the pulp is exposed in a small area or the remaining dentine is thin and close to the pulp, pulp capping is applied to maintain the vitality of the pulp. The ideal pulp capping material does not cause inflammation of the pulp, combines with the dentine without slight leakage, forms a dentin bridge, and is convenient for clinical use. In order to successfully perform pulp capping, various medicinal materials such as calcium hydroxide, MTA (Mineral Trioxide Aggregate), and adhesive resins are being studied, but among them, MTA is used for the most diverse purposes due to its bioaffinity. One of the important functions of direct pulp capping materials is to stimulate odontoblasts in the pulp and induce the biological effect of forming new dentin. This effect can achieve the sealing of the exposed pulp and prevent excessive nerve treatment. When performing nerve treatment, the pulp inside the tooth is completely removed, and it is ultimately difficult to preserve the natural tooth. Therefore, in order to improve overall oral health, it is also necessary to develop a direct pulp capping material that has an inducing function for the formation of dentin.

[0008] Resin restoration materials are mainly used, and resin restoration materials have excellent adhesion to tooth structure, but due to the toxicity of the monomer, patients occasionally feel uncomfortable after the restoration. In order to minimize the side effects of treatment, it is necessary to minimize the impact of the monomer on the pulp by sealing the exposed dentinal tubules and restoring the thickness of the damaged dentin. MTA, which is widely used as a pulp capping material, has excellent bioaffinity and sealing ability, but it will form a repaired dentin that is different from the original tubular dentin. In the long run, this repaired dentin will produce micro-leakage at the boundary with the original dentin. Summary of the invention

[0009] Technical issues

[0010] An object of the present invention is to provide a dentin adhesive that can prevent pulp necrosis, tooth removal or tooth loss that may occur when various dental restorative materials are applied to teeth.

[0011] Another object of the present invention is to provide a composition for covering the dental pulp. When the damaged area of ​​the tooth is close to the dental pulp or invades the dental pulp, and the exposed dental pulp area is small, the composition can be directly applied to seal the dental pulp for repair.

[0012] The objects of the present invention are not limited to the above-mentioned contents, and a person having ordinary knowledge in the technical field to which the present invention belongs can clearly understand other objects not mentioned from the following description.

[0013] Solution to the problem

[0014] In order to solve the above technical problems, according to one embodiment of the present invention, a composition for bonding dentin is provided, which comprises a peptide consisting of an amino acid sequence of the following general formula 1.

[0015] KY-R1-R2-R3-R4-R5-R6-R7-R8 (Formula 1)

[0016] In the general formula 1,

[0017] R1 is arginine (R), lysine (K) or glutamine (Q);

[0018] R2 is arginine (R) or glutamine (Q);

[0019] R3, R4 and R5 are arginine (R) or lysine (K), respectively;

[0020] R6 is asparagine (N) or serine (S); and

[0021] R7 and R8 are lysine (K) or tyrosine (Y).

[0022] Regarding dentin bonding, as long as the peptide can show an effect of promoting physiological mineralization of dentin, even a mutant peptide having a sequence different in one or more amino acid residues from the amino acid sequence constituting the peptide is included in the category of the peptide provided by the present invention.

[0023] Generally, the exchange of amino acids in proteins and polypeptides without changing the activity of the molecule as a whole is well known in the art to which the present invention belongs. The most common exchanges are exchanges 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, Asp / Gly. In addition, peptides may be included that increase the heat, pH, etc. related structural stability of the peptide or increase the ability to promote dentin synthesis through mutations or modifications in the amino acid sequence.

[0024] For example, even if glutamine, an acidic amino acid, located at position 3 of the peptide of sequence number 1 provided in the present invention is replaced by lysine or arginine, which are basic amino acids, the effect of the peptide provided in the present invention can be directly shown; even if arginine, a basic amino acid, located at position 4 or 5 of the peptide of sequence number 1 is replaced by glutamine, an acidic amino acid or lysine, which are basic amino acids, the effect of the peptide provided in the present invention can be directly shown; even if lysine, a basic amino acid, located at position 6, 7 or 9 of the peptide of sequence number 1 is replaced by arginine, a basic amino acid or tyrosine, which are aromatic amino acids, the effect of the peptide provided in the present invention can be directly shown; even if asparagine, an acidic amino acid, located at position 8 of the peptide of sequence number 1 is replaced by serine, which is a neutral amino acid, the effect of the peptide provided in the present invention can be directly shown; even if tyrosine, an aromatic amino acid, located at position 10 of the peptide of sequence number 1 is replaced by lysine, which are basic amino acids, the effect of the peptide provided in the present invention can be directly shown.

[0025] As described above, the acidic amino acids, basic amino acids or aromatic amino acids constituting the peptide of the present invention can directly exhibit the effects of the peptide provided in the present invention even if they are replaced by different acidic amino acids, basic amino acids, neutral amino acids or aromatic amino acids. Therefore, it is obvious that mutant peptides having a sequence different from that of one or more amino acid residues constituting the amino acid sequence of the peptide of the present invention also fall within the scope of the peptides provided in the present invention.

[0026] Furthermore, even if the peptide of the present invention has a form in which any amino acid is added to its N-terminus or C-terminus, the effect of the peptide provided in the present invention can be directly shown, and therefore it is included in the category of the peptide provided in the present invention. As one example, it can be 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 can be a form in which 1 to 100 amino acids are added to the N-terminus or C-terminus of the peptide, and as yet another example, it can be a form in which 1 to 24 amino acids are added to the N-terminus or C-terminus of the peptide.

[0027] Specifically, the peptides according to Formula 1 can be represented by Tables 1 to 12 below.

[0028] For example, a peptide (SEQ ID NO. 1) can be synthesized by the 9-fluorenylmethyloxycarbonyl (Fmoc) method, and the amino acids of the synthesized peptide are substituted to synthesize the peptides of each group (Tables 1 to 12).

[0029] N-KYQRRKKNKY-C (SEQ ID NO. 1)

[0030] First, the peptide of Group 1 is the peptide of Sequence No. 1, or is synthesized by substituting lysine or arginine for amino acids 5 to 7 of the peptide of Sequence No. 1 (Table 1).

[0031]

Table 1

[0032]

[0033]

[0034] Then, the amino acids at positions 5 to 7 of the peptide of SEQ ID NO: 1 were substituted with lysine or arginine, and the amino acid at position 8 was substituted with serine, thereby synthesizing peptides of Group 2 (Table 2).

[0035]

Table 2

[0036]

[0037] Then, the amino acids at positions 5 to 7 of the peptide of SEQ ID NO: 1 were substituted with lysine or arginine, and the amino acid at position 9 was substituted with tyrosine, thereby synthesizing peptides of Group 3 (Table 3).

[0038]

Table 3

[0039]

[0040]

[0041] Then, the amino acids at positions 5 to 7 of the peptide of sequence number 1 were replaced with lysine or arginine, the amino acid at position 8 was replaced with serine, the amino acid at position 9 was replaced with tyrosine, and the amino acid at position 10 was replaced with lysine, thereby synthesizing peptides of group 4 (Table 4).

[0042]

Table 4

[0043]

[0044] Then, the amino acid at position 3 of the peptide of sequence number 1 was substituted with arginine, the amino acid at position 4 was substituted with glutamine, and the amino acids at positions 5 to 7 were substituted with lysine or arginine, thereby synthesizing peptides of group 5 (Table 5).

[0045]

Table 5

[0046]

[0047]

[0048] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced with arginine, the amino acid at position 4 was replaced with glutamine, the amino acids at positions 5 to 7 were replaced with lysine or arginine, and the amino acid at position 8 was replaced with serine, thereby synthesizing peptides of group 6 (Table 6).

[0049]

Table 6

[0050]

[0051] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by arginine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 7 (Table 7).

[0052]

Table 7

[0053]

[0054]

[0055] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by arginine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 8 was replaced by serine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 8 (Table 8).

[0056]

Table 8

[0057]

[0058] Then, the amino acid at position 3 of the peptide of sequence number 1 was substituted with lysine, the amino acid at position 4 was substituted with glutamine, and the amino acids at positions 5 to 7 were substituted with lysine or arginine, thereby synthesizing peptides of group 9 (Table 9).

[0059]

Table 9

[0060]

[0061] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by lysine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, and the amino acid at position 8 was replaced by serine, thereby synthesizing peptides of group 10 (Table 10).

[0062]

Table 10

[0063]

[0064] Then, the amino acid at position 3 of the peptide of sequence number 1 was replaced by lysine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 11 (Table 11).

[0065]

Table 11

[0066]

[0067] Finally, the amino acid at position 3 of the peptide of sequence number 1 was replaced by lysine, the amino acid at position 4 was replaced by glutamine, the amino acids at positions 5 to 7 were replaced by lysine or arginine, the amino acid at position 8 was replaced by serine, the amino acid at position 9 was replaced by tyrosine, and the amino acid at position 10 was replaced by lysine, thereby synthesizing peptides of group 12 (Table 12).

[0068]

Table 12

[0069]

[0070]

[0071] As another embodiment of the present invention, the present invention provides a polynucleotide encoding the peptide.

[0072] For the polynucleotide, more than one base can be mutated by substitution, knockout, insertion or a combination thereof. In the case of preparing by chemically synthesizing a nucleotide sequence, a synthesis method known in the art to which the present invention belongs can be used, such as the method described in the literature (Engels and Uhlmann, Angew Chem Int Ed Engl., 37: 73-127, 1988), and can be synthesized using triester, phosphite, phosphoramidite and H-phosphate methods, polymerase chain reaction (PCR) and other automatic primer methods, oligonucleotide synthesis on a solid support, etc. For example, the polynucleotide encoding the peptide of the present invention may include the base sequence of sequence number 4.

[0073] As another embodiment, the present invention provides an expression vector comprising the polynucleotide, a transformant comprising the expression vector, and a method for producing the peptide using the transformant.

[0074] The term "expression vector" of the present invention refers to a recombinant vector that can express a target peptide in a target host cell, and means a gene construct that contains necessary regulatory elements that are operably linked to express a gene insert. The expression vector contains expression regulatory elements such as a start codon, a stop codon, a promoter, and an operator gene. The start codon and the stop codon are usually considered to be part of the nucleotide sequence encoding the polypeptide, and must exhibit an effect in an individual when the gene construct is administered, and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.

[0075] The term "operably linked" of the present invention 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 in order to perform a common function. For example, operably linking a promoter and a nucleic acid sequence encoding a protein or RNA can affect the expression of the coding sequence. Operable linkage with an expression vector can be prepared using gene recombination techniques known in the art to which the present invention belongs, and site-specific DNA cleavage and ligation can use enzymes commonly known in the art to which the present invention belongs, etc.

[0076] In addition, in order to promote the separation of peptides from the cell culture fluid, the expression vector may include a signal sequence for discharging the peptides. Specific initiation signals may also be required when performing effective translation of the inserted nucleotide sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, an exogenous translation regulation signal that may include the ATG initiation codon may be provided. These exogenous translation regulation signals and initiation codons may be a variety of natural and synthetic sources. Expression efficiency may be increased by introducing appropriate transcription or translation enhancers.

[0077] Meanwhile, in order to facilitate the detection of the peptide, the expression vector may further comprise a protein tag which can be optionally removed using an endopeptidase.

[0078] The term "tag" of the present invention refers to a molecule that exhibits an activity or characteristic that can be quantified, and may be a fluorescent molecule including a chemical fluorescent substance (fluoracer) (such as fluorescein) and a polypeptide fluorescent substance (such as fluorescent protein (GFP) or related proteins); or an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, a streptavidin tag, etc. In particular, when an epitope tag is used, a peptide tag consisting of 6 or more amino acid residues may be preferably used, and a peptide tag consisting of 8 to 50 amino acid residues may be more preferably used.

[0079] 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 the physiological mineralization of dentin. The vector used in this case may preferably be a plasmid DNA, a phage DNA, etc., and more preferably may be a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), an Escherichia coli-derived plasmid (pYG601 BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (pUB110, pTP5, etc.), a yeast-derived plasmid (YEp13, YEp24, YCp50, etc.), a phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (retrovirus, adenovirus, vaccinia virus, etc.) virus, etc.), insect virus vectors (baculovirus, etc.), but are not particularly limited thereto as long as the peptide can be produced. The expression vector presents different protein expression amounts and modifications depending on the host cell, so it is preferred to select and use the host cell that best suits the purpose.

[0080] The expression vector provided in the present invention can be introduced into the host for transformation, thereby preparing the transformant provided in the present invention, and the polynucleotide contained in the expression vector can be expressed to produce the peptide. The transformation can be carried out by various methods, such as using CaCl 2 Precipitation method, in CaCl 2 The precipitation method includes the Hanahan method which uses a reducing substance called dimethyl sulfoxide (DMSO (dimethyl sulfoxide)) to improve efficiency, electroporation, calcium phosphate precipitation, protoplast fusion, stirring method using silicon carbide fiber, Agrobacterium-mediated transformation method, transformation method using polyethylene glycol (PEG), dextran sulfate, liposome and drying / inhibition-mediated transformation method, but is not particularly limited thereto as long as the peptide can be produced. Furthermore, the host used in the preparation of the transformant is not particularly limited as long as it can produce the peptide, but may be bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc.; yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc.; fungal cells such as Pichia pastoris, etc.; insect cells such as Drosophila, Spodoptera Sf9 cells, etc.; animal cells such as CHO cells, COS cells, NSO cells, 293 cells, human melanoma cells, etc.; or plant cells.

[0081] The transformant can also be used in the method for producing the peptide of the present invention that provides the effect of promoting the physiological mineralization of dentin. Specifically, the method for producing the peptide of the present invention that provides the effect of promoting the physiological mineralization of dentin may include: step (a), obtaining a culture by culturing the transformant; and step (b), recovering the peptide of the present invention from the culture.

[0082] The term "culture" of the present invention means a method of growing and developing microorganisms under appropriately artificially adjusted environmental conditions. In the present invention, the method of culturing the transformant can be carried out using a method widely known in the technical field to which the present invention belongs. Specifically, with respect to the culture, continuous culture can be carried out in a batch process or a fedbatch or repeated fed batch process, but as long as it can be produced by expressing the peptide of the present invention that provides the effect of promoting the physiological mineralization of dentin, it is not particularly limited thereto.

[0083] The culture medium used for the culture needs to be a conventional culture medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, etc., under aerobic conditions, and the temperature, pH, etc., and the conditions of the specific strain need to be adjusted in an appropriate manner. As a usable carbon source, a mixed sugar of glucose and xylose is used as the main carbon source, in addition to: sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, cellulose; and oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc.; and fatty acids such as palmitic acid, stearic acid, linoleic acid; and alcohols such as glycerol, ethanol; and organic acids such as acetic acid. These substances can be used individually or as a mixture. As usable nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used; amino acids such as glutamic acid, methionine, glutamine; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. can be used. These nitrogen sources can be used alone or in combination. In the culture medium, as a phosphorus source, monopotassium phosphate, dipotassium phosphate and corresponding sodium salts can be included. As usable phosphorus sources, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium salts can be included. And as inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate and calcium carbonate can be used. Finally, growth essential substances such as amino acids and vitamins can be used on the basis of the substances.

[0084] In addition, appropriate precursors may be used in the culture medium. The raw materials may be added to the culture in a batch, fed-batch or continuous manner during the culture process, but are not particularly limited thereto. The pH of the culture may be adjusted by using an alkaline compound such as sodium hydroxide, potassium hydroxide, ammonia, or the like; an acidic compound such as phosphoric acid or sulfuric acid, or the like, in an appropriate manner.

[0085] Furthermore, the generation of bubbles can be suppressed by using defoamers such as fatty acid polyethylene glycol esters. In order to maintain a good aerobic state, oxygen or 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. And the culture is continued until the production amount of the peptide reaches the maximum. For this purpose, it can usually be achieved within 10 hours to 100 hours.

[0086] Furthermore, the step of recovering the peptide from the culture may be performed by a method known in the art to which the present invention belongs. Specifically, the recovery method is not particularly limited as long as it is applicable to the recovery of the produced peptide, and preferably, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) and the like can be used.

[0087] The term "prevention" of the present invention means all actions to inhibit or delay pulp necrosis, tooth removal or tooth loss, or secondary caries or pulpitis caused by dental restorative materials by administering a composition for bonding dentin and a composition for covering dental pulp comprising the peptide of the present invention.

[0088] The term "treatment" of the present invention means all actions of performing tooth restoration treatment by administering a restorative material comprising a composition for bonding dentin, a composition for bonding dentin, or a composition for covering a dental pulp to an individual in need of tooth restoration treatment, thereby promoting the physiological remineralization of dentin between the tooth and the restorative material, wherein the composition for bonding dentin comprises the peptide of the present invention as an active ingredient.

[0089] The composition of the present invention can be prepared into the form of a composition for treating damaged tooth substance such as caries, fractures, etc., which further includes an appropriate carrier (natural or non-natural carrier), excipient or diluent commonly used in the preparation of the composition for restoring the tooth. Specifically, the composition can be formulated into a sterile injection that can be administered to the damage-inducing site of the tooth substance such as caries, fractures, etc. according to the usual method. In the present invention, the carrier, excipient and diluent that can be included in the composition can be exemplified by lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, collagen, etc. When preparing the formulation, commonly used diluents or excipients such as fillers, brighteners, binders, wetting agents, disintegrants, surfactants, etc. can be used for preparation. In particular, it can include sterile aqueous solutions, non-aqueous solvents, suspending agents, emulsions, freeze-dried agents, suppositories, ointments (for example, endodontic transplant materials, etc.), etc. As non-aqueous solvents and suspending agents, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, etc. can be used; injectable esters such as ethyl oleate, etc. can be used. As the base of the suppository, synthetic fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, lauric acid glyceride fat, glycerin gelatin, etc. can be used.

[0090] The content of the peptide contained in the composition of the present invention may be 0.0001 wt % to 50 wt %, more preferably 0.01 to 20 wt %, based on the total weight of the final composition, but is not limited thereto.

[0091] The composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention refers to an amount sufficient for the treatment or prevention of a disease at a reasonable benefit / risk ratio that can be applied to medical treatment or prevention. The effective dosage level can be determined based on the severity of the disease, the activity of the drug, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the administration time, administration route and excretion rate of the composition of the present invention used, the treatment time, the elements of the drug used in combination with or containing the composition of the present invention, and other elements known in the medical field. The composition of the present invention can be administered independently or in combination with a known composition for treating damaged tooth tissue such as caries, fractures, etc. It is important to take all the above factors into consideration and administer the drug in an amount that has no side effects and can achieve the maximum effect with the minimum amount.

[0092] A person skilled in the art of the present invention may determine the dosage of the composition of the present invention by considering the purpose of use, the degree of disease poisoning, the age, weight, sex, previous illness of the patient, or the type of substance used as an active ingredient. For example, each adult may be administered about 0.1 ng to about 100 mg / kg of the composition of the present invention, preferably, it can be administered at 1 ng to about 10 mg / kg, and the administration frequency of the composition of the present invention may be once a day or divided into doses for multiple administrations, but is not particularly limited thereto. The dosage does not limit the scope of the present invention in any aspect.

[0093] As another embodiment, the present invention provides a method for treating damaged tooth such as caries or fractures, comprising the step of administering the composition in a pharmaceutically effective amount to an individual suffering from damaged tooth such as caries or fractures.

[0094] The term "subject" of the present invention is intended to include without limitation mammals and the like that require treatment of damaged tooth such as caries and fractures, including mice and livestock, but humans may be excluded from subjects suffering from the disease.

[0095] The administration route of the composition for treating damaged tooth such as caries and fractures of the present invention can also be administered by any common route as long as the target tissue can be reached. The composition of the present invention can provide all dosage forms suitable for local application according to the purpose, but is not particularly limited thereto. For example, it can be administered by oral, transdermal, intravenous, intramuscular, or subcutaneous injection. The composition can be an injection, a skin external solution, a suspension, an emulsion, a gel, a patch or a spray, but is not limited thereto. The dosage form can be easily prepared by the usual methods in the art, and surfactants, excipients, wettable powders, emulsifying agents, suspending agents, salts or buffers for controlling osmotic pressure, colorants, spices, stabilizers, preservatives, preservatives or other commonly used adjuvants can be appropriately used.

[0096] As another embodiment, the present invention provides a quasi-drug composition for preventing or improving damaged tooth such as caries and fractures, comprising the peptide.

[0097] The term "improvement" according to the present invention is intended to refer to a parameter related to the state being treated, for example, all actions that at least reduce the degree of symptoms.

[0098] In the present invention, the improvement can be explained as follows: a composition comprising the peptide of the present invention as an active ingredient is administered to an individual who needs to treat damaged tooth such as caries, fractures, etc., to promote the synthesis of dentin, thereby improving the symptoms of damaged tooth such as caries, fractures, etc. or obtaining favorable changes.

[0099] The term "quasi-drugs" in the present invention means items that are used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases in humans or animals and have a milder effect than drugs. For example, according to the Pharmaceutical Affairs Law, quasi-drugs refer to items other than items used as drugs, including fiber / rubber products used to treat or prevent human / animal diseases, items that are not instruments or machines and similar items that have a mild or no direct effect on the human body, sterilizers / insecticides used to prevent infectious diseases, etc.

[0100] In the present invention, the type and dosage form of the quasi-drug composition containing the peptide are not particularly limited, but as an example, it can be a disinfectant cleanser for skin or hair, a skin or hair cleansing product, a soap, a shampoo, a skin ointment, etc.

[0101] As another embodiment, the present invention provides a health functional food composition for preventing or improving damaged tooth such as caries and fractures, comprising the peptide.

[0102] The term "food" of the present invention includes meat, sausage, bread, chocolate, candy, fast food, biscuits, pizza, instant noodles, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, rehydration, alcoholic beverages, vitamin complexes, health functional foods and health foods, etc., including all foods in the conventional sense.

[0103] The functional food is the same term as food for special health use (FoSHU), which refers to a food with high medical and medical effects that can be processed to effectively present the regulating function of the organism in addition to providing nutrition. Among them, the so-called "function" means to regulate nutrients (to the structure and function of the human body) or to achieve favorable effects in health purposes (such as physiological effects, etc.). The food of the present invention can be prepared by the method commonly used in the technical field to which the present invention belongs, and the raw materials and ingredients that are usually added in the technical field can be added to prepare it during the preparation. In addition, as for the dosage form of the food, as long as it is a dosage form that can be identified as food, it can be prepared without restriction. The food composition of the present invention has the following advantages: it can be prepared into a variety of dosage forms, and unlike ordinary medicines, food can be used as a raw material, so there are no side effects that may occur when taking medicines for a long time, and it is excellent in portability, so the food of the present invention can be taken as an auxiliary agent for enhancing the effect of preventing or improving the damaged tooth substance such as caries and fractures.

[0104] The health food refers to a food that has a positive effect of maintaining or improving health compared to ordinary food, and the health supplement food refers to a food for health supplement purposes. Depending on the situation, the terms health functional food, health food, and health supplement food can be used interchangeably.

[0105] Specifically, the health functional food refers to a food prepared by adding the peptide of the present invention to food materials such as beverages, teas, spices, chewing gums, biscuits, etc., or preparing the food in the form of encapsulation, powder, suspension, etc. When the food is ingested, it brings specific effects on health. However, unlike ordinary medicines, since food is used as the raw material, the advantage is that there are no side effects that may occur when taking medicines for a long time.

[0106] The food composition of the present invention can be ingested daily, and therefore can be expected to have a good effect in preventing or improving damaged tooth tissue such as caries and fractures, and can therefore be used very effectively.

[0107] The food composition may further include a physiologically acceptable carrier. The type of the carrier is not particularly limited, and any carrier commonly used in the technical field to which the present invention belongs may be used.

[0108] Furthermore, the food composition may include additives that can be commonly used in food compositions to improve smell, taste, vision, etc. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folic acid, pantothenic acid, etc. may be included. Furthermore, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc. may be included. Furthermore, amino acids such as lysine, tryptophan, cysteine, valine, etc. may be included.

[0109] Moreover, the food composition may contain food additives, such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and high-efficiency bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar pigments, etc.), colorants (sodium nitrite, sodium acetate, etc.), bleaching agents (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (sweetness, cyclamates, saccharin, sodium, etc.), spices (vanillin, lactones, etc.), expanders (alum, D-potassium hydrogen tartrate, etc.), strengtheners, emulsifiers, thickeners (paste), film-forming agents, glue bases, foam inhibitors, solvents, improvers, etc. The additives may be selected and used in appropriate amounts according to the type of food.

[0110] Peptide of the present invention can be directly added or used together with other foods or food ingredients, and can be suitably used according to conventional methods.Can suitably determine the mixed amount of active ingredient according to its purpose of use (prevention, health or therapeutic treatment).Usually, when preparing food or beverage, relative to food or beverage, food composition of the present invention can add below 50 weight portions, specifically, can add the amount below 20 weight portions.But, in the case of long-term intake for the purpose of health and hygiene, can comprise the amount below the described scope, owing to there is no problem aspect safety, can also use the amount above the described scope as active ingredient.

[0111] As an example of the food composition of the present invention, it can be used as a health drink composition. In the case, it can contain a variety of flavoring agents or natural carbohydrates as added ingredients, just like general beverages. The natural carbohydrates can be monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose; polysaccharides, such as dextrin and cyclodextrin; sugar alcohols, such as xylitol, sorbitol, erythritol, etc. Sweeteners can use natural sweeteners, such as thaumatin and stevia extract; synthetic sweeteners, such as saccharin and aspartame, etc. The ratio of the natural carbohydrates in each 100 mL of the health drink composition of the present invention can generally be about 0.01g to 0.04g, specifically about 0.02g to 0.03g.

[0112] In addition to the above, the health drink composition can include multiple nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectin acid, pectinate, alginic acid, alginate, organic acid, protective colloid thickener, pH adjusting agent, stabilizer, preservative, glycerine, ethanol or carbonating agent etc. In addition, the pulp used to prepare natural fruit juice, fruit juice beverage or vegetable beverage can be included. This composition can be used independently or in combination. Although the ratio of this additive is not particularly important, the health drink composition of the present invention of every 100 weight portions is selected usually in the range of 0.01 to 0.1 weight portion.

[0113] As for the food composition of the present invention, if it can show the effect of preventing or improving damaged tooth substance such as caries and fractures, the peptide of the present invention can be included in various weight %; specifically, based on the total weight of the food composition, it can contain 0.00001 to 100 weight % or 0.01 to 80 weight % of the peptide of the present invention, but it is not limited to this.

[0114] Effects of the Invention

[0115] A dentin adhesive according to an embodiment of the present invention can prevent or inhibit secondary caries or pulpitis caused by 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.

[0116] According to an embodiment of the present invention, a composition for covering the dental pulp is directly applied to seal the dental pulp and repair it when the damage range of the tooth is close to the dental pulp or invades the dental pulp, and the exposed dental pulp is small, thereby achieving the sealing of the dental pulp, thereby preventing or inhibiting the slight leakage that may occur at the boundary of the repaired dentin, thereby providing high reliability and durability for the treatment of tooth damage.

[0117] The effects of the present invention are not limited to the above-mentioned contents, and a person having ordinary knowledge in the technical field to which the present invention belongs can clearly understand other effects not mentioned from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 This is the effect of a mixed solution of dentin adhesive and KH001 on the expression of odontogenic differentiation marker genes of human dental pulp cells. The marker genes are DSPP, DMP1, and BSP, and the expression of the genes was measured by RT-PCR. All values ​​of the following results are expressed as the average and standard deviation of the experimental results of three times, and *p<0.05 is the result compared with the control group.

[0119] Figure 2The results of measuring the transmittance of dentinal tubules to determine whether KH001 successfully reaches the pulp when treating a human tooth with exposed dentin with a mixed solution of dentin adhesive and KH001. A is a schematic diagram of the sample preparation process for measuring the transmittance of dentinal tubules, and BD are microscopic images taken with a confocal microscope.

[0120] Figure 3 The results of histological evaluation of the regeneration ability of tubular dentin in a mixed solution of dentin adhesive and KH001. After making a dish-shaped injury on the cervical part of the tooth of a beagle dog to expose the dentin, a dentin adhesive without KH001 was applied to the control group and then repaired with resin, while a dentin adhesive containing KH001 was applied to the experimental group and then repaired with resin. In order to investigate the formation of tubular dentin, a histological evaluation was performed by hematoxylin / eosin staining (H&Estaining), and the results were obtained. The dotted line shows the boundary of the newly formed tubular dentin, and TD (tertiary dentin) refers to tubular dentin.

[0121] Figure 4 The results are the results of observing the expression of odontogenic differentiation marker genes in human dental pulp cells under MTA conditioned medium at different concentrations of KH001. The marker genes are DSPP, DMP1, and BSP, and the gene expression was measured by RT-PCR. All values ​​of the following results are expressed as the average and standard deviation of the experimental results of three times, and *p<0.05, **p<0.01 are compared with the control group.

[0122] FIG. 5 shows the results of observing the expression changes of odontoblast differentiation gene markers based on the differentiation date in an experiment of human dental pulp cells treated with MTA-conditioned media to confirm the efficacy of KH001.

[0123] Figure 6 The purpose of this study was to confirm the efficacy of KH001 in an experiment with human dental pulp cells treated with MTA-conditioned media and to observe the changes in odontoblast differentiation and mineralization capacity.

[0124] Figure 7 The purpose of this study was to confirm the efficacy of KH001 in an experiment with human dental pulp cells treated with MTA-conditioned media and to observe changes in wound healing ability and cell migration ability.

[0125] Figure 8 These are photographs taken with a confocal microscope in order to observe the penetrating ability of the composition for covering dental pulp according to an example of the present invention into the dentinal tubules of the dentin.

[0126] Fig. 9 The present invention is a schematic diagram showing the site to which the composition for bonding dentin and the composition for covering dental pulp are applied in the process of treating a damaged tooth and the states before and after the application. DETAILED DESCRIPTION

[0127] If you refer to the following and Figure 1 The purpose and effect of the present invention and the technical structure used to achieve the same will be clear from the detailed description of the embodiments. In describing the present invention, when it is judged that the detailed description of the known function or structure may unnecessarily confuse the gist of the present invention, the detailed description will be omitted. Moreover, the terms described below, as terms defined in consideration of the functions in the present invention, may vary according to the intention or convention of the user or operator.

[0128] However, the present invention is not limited to the embodiments disclosed below, and can be embodied in various forms that are different from each other. However, this embodiment is provided to make the disclosure of the present invention more complete and to fully explain the scope of the invention to people with ordinary knowledge in the technical field to which the present invention belongs, and the present invention is only defined by the scope of the claims. Therefore, this definition should be made based on the entire content of this specification.

[0129] The following is a detailed description of the embodiments of the present invention.

[0130] Example 1: Materials and Methods

[0131] Example 1-1: Preparation of peptides

[0132] The KH001 peptide (SEQ ID NO: 96) is composed of a synthetic peptide corresponding to the 10 amino acid residues 344-353 fragment (KYKQKRRSYK) of the hCPNE7 protein. The peptide KH001 was synthesized using a solid phase method based on 9-fluorenylmethyloxycarbonyl (Fmoc), and the purity of the synthesized peptide KH001 was measured by high performance liquid chromatography and was above 97%.

[0133] Example 1-2: Cell culture

[0134] 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's medium (DMEM; Gibco BRL., Carlsbad, CA, USA), and 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 in 5% CO 2 The cells were incubated at 37°C under an atmosphere supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL) and antibiotic-antimycotic (Gibco BRL).

[0135] For use in vitro and ex vivo experiments, cells were cultured in Minimum Essential Medium α (MEM-α; Gibco BRL). For differentiation of human dental pulp cells (hDPC) and MDPC-23 cells, 80-90% confluent cells were cultured for 3 weeks in the respective medium supplemented with 5% FBS, ascorbic acid (50 μg / mL) and β-glycerophosphate (10 mM). Passages 2 to 4 were used for hDPSCs, and passages 23 to 25 were used for MDPC-23 cells.

[0136] Example 2-1-1: Preparation of a composition for bonding dentin

[0137] Combine KH001 with Bisco Dental TM The company's dentin adhesive ABU (All-Bond Universal) is prepared by adding 2 mg of KH001 to every 1 mL of ABU, and stirring with a stirrer for about 40 minutes under reduced pressure conditions (-750 mmHg) (stirring conditions: slurry (PADDLE) is 10-30 rpm, dispersion (DISPERSE) is 500-600 rpm, high speed (HOMO) is 2400-3200 rpm). Then store in a light-proof container. The prepared composition for bonding dentin is refrigerated and stored at 4°C for about 3 months after preparation, and is applicable to Examples 2-3 and 2-4 and is recorded in Table 13.

[0138] Example 2-1-2: Preparation of a composition for bonding dentin

[0139] In the cell experiment of human dental pulp cells (hDPC) prepared in Example 1-2, in order to prevent the side effects such as cell death caused by the monomer toxicity of the dentin adhesive, ABU was diluted at a ratio of 1:5000 in 10% α-MEM and used to prepare a composition for bonding dentin. KH001 peptide powder was dissolved in the diluent to prepare it in a manner that it contained 5ug / ml and 10ug / ml respectively, and the composition for bonding dentin prepared in this way was applied to Example 2-2. After preparation, the prepared composition for bonding dentin was refrigerated and stored at 4°C.

[0140] Example 2-2: Expression regulation analysis of odontoblast differentiation marker genes

[0141] After the human dental pulp cells (hDPCs) prepared in Example 1-2 were treated with the composition for bonding dentin of Example 2-1-2, the expression of DSPP, DMP1, and BSP genes was measured by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analysis. Figure 1 The gene expression values ​​shown in the middle graph are expressed as the average and standard deviation of the experimental results of three times, and *p<0.05 and **p<0.01 are the results compared with the control group.

[0142] refer to Figure 1 As the concentration of KH001 increased, the expression levels of DSPP, BSP, and DMP, which are marker genes for odontoblast differentiation, increased. When KH001 was treated at a concentration of 10ug / ml, the expression of DSPP, DMP1, and BSP mRNA increased by more than 2.5 times.

[0143] Example 2-3: Observation of light transmittance of dentinal tubules

[0144] The extracted human teeth were treated with the composition for bonding dentin prepared by Example 2-1-1 of the present invention and the transmittance of the dentinal tubules was observed. The extracted human teeth were impacted human third molars of patients between the ages of 18 and 22 provided by Seoul National University Dental Hospital. The experimental protocol was approved by the Institutional Review Board (IRB number: S-D20140007), and consent was obtained from all patients in advance.

[0145] The crown of the extracted human tooth was cross-cut with a diamond saw to expose the dentinal tubules, and then the exposed human tooth was treated. The composition for bonding dentin prepared in Example 2-1-1, which contained KH001 peptide (SEQ ID NO: 96) labeled with rhodamine as a fluorescent staining agent, was applied to the crown surface of the extracted human tooth.

[0146] The results are as follows: the dentin bonding composition was applied without a brush application process or an air drying process, and then left to stand for about 30 seconds for photopolymerization, and then a confocal image was taken.

[0147] Figure 2 The results are the results of measuring the light transmittance of the dentinal tubules in which KH001 successfully reaches the pulp when a mixed solution of dentin adhesive and KH001 is treated for about 30 seconds in a human tooth with exposed dentin. Figure 2 , A is a schematic diagram of the sample preparation process for measuring the transmittance of dentinal tubules. Figure 2 BD is a microscopic photograph taken with a confocal microscope, and the observation results confirmed that the peptide KH001 flowed smoothly into the pulp cavity along the dentinal tubules.

[0148] Example 2-4. Histological evaluation of the regenerative capacity of tubular dentin

[0149] After creating a dish-shaped injury at the cervical region of the teeth of beagle dogs to expose the dentin, a dentin adhesive not mixed with peptide KH001 was applied to the control group and then restored with resin, while a dentin adhesive containing KH001 was applied to the experimental group and then restored with resin. Histological evaluation was then performed by hematoxylin / eosin staining (H&Estaining) to investigate the formation of tubular dentin.

[0150] Figure 3 These are the results of histological evaluation of the tubular dentin regeneration ability of the dentin bonding composition of Example 2-1-1. Figure 3 , the dotted line shows the boundary of the newly formed tubular dentin, and TD (newly formed tertiary dentin) is the dentin with newly formed dentinal tubules.

[0151] refer to Figure 3In the control group (I) where only the dentin adhesive (ABU) was applied, no tubular dentin was formed below the damaged dentin site, but in the experimental group (II) (including 5ug / ml of KH001) and the experimental group (III) (including 10ug / ml of KH001) where the composition for bonding dentin using KH001 prepared in Example 2-1-2 was applied, tubular dentin was confirmed to be formed on the pulp side below the damaged dentin site in the experimental group (II) (including 5ug / ml of KH001) and the experimental group (III) (including 10ug / ml of KH001). In addition, in the pulp adjacent to the newly formed tubular dentin, odontoblasts were arranged in a palisade shape, indicating that it was an activated healthy pulp.

[0152] Table 13 is the result of a shear bond strength test using the Ultradent method to confirm the bonding strength between the composition for bonding dentin containing KH001 prepared by Example 2-1-1 and the tooth. For the dentin, the surface was polished with 320-grit sandpaper and 600-grit sandpaper, and then ABU of Bisco Company as a control group and a dentin adhesive containing KH001 at a concentration of 2 mg / mL as an experimental group were applied in a self-etching manner for about 10 to 15 seconds, and then photopolymerized with a light source of 1000 mW / cm2 intensity, and then scrubbed, and irradiated with a light source of the same intensity Duo-Link Universal TM (DLU, Bisco) for 20 seconds and bonded. The samples were then stored in pure water (DI water) at 37°C and the results were measured. Each group contained 10 samples.

[0153]

Table 13

[0154] Adhesive+Cement SBS,Mpa(SD) ABU+DLU 34.20(8.22) ABU w / KH001+DLU 33.43(8.36)

[0155] Table 13 shows the bonding strength of the composition for bonding dentin prepared by Example 2-1-1. Referring to Table 13, even if KH001 is contained at a high concentration of about 200 times the effective concentration, the bonding strength of the composition for bonding dentin provides a similar level of bonding strength compared to the existing dentin adhesive. From this result, it can be seen that even if KH001 is contained in the composition for bonding dentin at a concentration of about 2 mg / mL or less, it can provide the same level of bonding strength as the conventional dentin adhesive.

[0156] Example 3-1: Preparation of a composition for covering dental pulp

[0157] For MTA (mineral trioxide agglomerates) (ProRoot MTA; Dentsply Sirona, New York, PA, USA), the powder / liquid ratio was prepared according to the manufacturer's instructions and mixed using a metal slab on a sterilized glass plate and then cured at room temperature for 1 hour for complete curing.

[0158] After solidification, MTA was ground into powder in a sterilized mortar, immersed in a-MEM (MEM-α; Gibco BRL), a culture medium for human dental pulp cells, for 24 hours and stored at about 37° C. to prepare a conditioned media.

[0159] Then, KH001 was treated at a concentration of 5 ug / mL and 10 ug / mL in a conditioned medium diluted at a ratio of x1 / 5, respectively, to prepare a composition for covering a dental pulp.

[0160] Example 3-2: Evaluation of the expression levels of odontogenic differentiation marker genes (DSPP, DMP1, BSP) and CPNE7 genes in the composition for covering dental pulp according to different concentrations of KH001

[0161] Using Tri-Reagent TM Total RNA was extracted from human dental pulp cells and then treated with reverse transcriptase and oligo(dT) primer (Invitrogen) to synthesize cDNA.

[0162] For the gene amplification process, 1 μL of the reverse transcribed cDNA was mixed with primers corresponding to each gene and SYBR Green Supermix in a 96-well plate, and then a quick cycle was performed according to the manufacturer's instructions.

[0163] The amount of PCR product was measured using the ratio to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the relative expression levels were compared using the CT (comparative threshold cycle) method.

[0164] Figure 4The results of measuring the expression of odontogenic differentiation marker genes and CPNE7 gene expression in human dental pulp cells according to different concentrations of KH001 (KH001 in MTA conditioned medium) of the composition for covering dental pulp by RT-PCR. All the results are expressed as the mean and standard deviation of the experimental results of three times, *p<0.05, **p<0.01 are the results compared with the control group.

[0165] Example 3-3: Evaluation of the differentiation expression levels of odontoblast differentiation marker genes (DSPP, DMP1, BSP) and CPNE7 genes according to the date of treatment with the composition for covering dental pulp (10 ug / ml KH001 under MTA-conditioned medium conditions)

[0166] In order to compare the function of the composition for covering dental pulp comprising 10ug / ml of KH001 prepared in Example 3-1 in inducing differentiation of human dental pulp cells into odontoblasts, a control group (Ctrl) treated with differentiation medium only, a group treated with MTA-conditioned differentiation medium (MTA), and a composition for covering dental pulp (a group treated with MTA-conditioned / CPNE7 functional peptide together, MTA+KHOO1) were compared.

[0167] The composition of the differentiation medium was prepared by basically adding 1% antibiotics, 10 mM β-glycerophosphate, and 50 μg / mL ascorbic acid to α-MEM supplemented with 5% FBS.

[0168] After human dental pulp cells were treated with each culture medium, RT-PCR was performed to observe the expression changes of odontoblast differentiation gene markers on day 0, day 7, and day 14.

[0169] Figure 5 is the result of observing the expression changes of differentiation gene markers of odontoblasts based on the differentiation date in order to confirm the efficacy of KH001 in an experiment of human dental pulp cells treated with MTA-conditioned media (MTA+KH001). MTA is a preparation suitable for use when the pulp is exposed or when the pulp is not exposed but only a thin thickness of dentin is left and the pulp is expected to be damaged. Therefore, the odontoblasts originally present in the exposed pulp part are likely to die, and in order to restore them and form the third stage of dentin in the exposed pulp part, it is important to induce the differentiation of pulp cells into odontoblasts.

[0170] Referring to Figure 5, during the differentiation of human dental pulp cells, when observing the expression of odontoblast markers and mineralization-related genes, the expression levels of DMP1 and CPNE7 in the group treated with MTA and CPNE7 functional peptides on the 7th day of differentiation were significantly increased compared to the group treated with MTA alone. However, on the 14th day, the expression of DSPP, DMP1, and CPNE7 genes was observed to be significantly higher in the group treated with MTA and CPNE7 functional peptides than in the group treated with MTA alone. BSP, a gene related to the formation of bone-like dentin, was observed to have no significant increase on both the 7th and 14th days compared to the group treated with MTA alone, and the expression level was equivalent to that of the case where MTA alone was treated.

[0171] Example 3-4: Evaluation of differentiation and mineralization capacity of odontoblasts treated with MTA-conditioned medium

[0172] In order to confirm the effect of CPNE7 functional peptide on the differentiation and mineralization ability of odontoblasts in the presence of MTA, Alizarin red staining (ARS) was performed.

[0173] In a 6-well plate, 1×10 5 After human dental pulp cells (passage number 2-4) were inoculated at a density of cells / well, when the confluency reached 80%, the cells were treated with differentiation medium corresponding to the control group and the experimental group for 7, 14, and 21 days, and the differentiation medium was replaced every 2 days.

[0174] To fix the cells, they were treated with 4% paraformaldehyde at 4°C for 15 minutes, and the minerals were stained by treating with 40 mM Alizarin red S at room temperature for 30 minutes using an Alizarin red S staining Quantification kit.

[0175] Thereafter, for quantitative analysis, the mixture was treated with 10% acetic acid at room temperature for 30 minutes and then centrifuged to extract 500 μL of a supernatant, to which 200 μL of 10% ammonium hydroxide was further added for neutralization.

[0176] Afterwards, the absorbance was measured at 405 nm together with Alizarin Red S standard solution (ARS standard). All experiments were performed in triplicate.

[0177] Figure 6The purpose of this study was to confirm the efficacy of KH001 (a composition for covering dental pulp) in an experiment using human dental pulp cells treated with MTA-conditioned media and to observe changes in odontoblast differentiation and mineralization ability.

[0178] The clinical situation in which MTA is applicable is when pulp exposure leads to death of odontoblasts. In this case, in order to maintain the pulp, in addition to inducing pulp cells to differentiate into odontoblasts, it is also necessary for differentiated odontoblasts to effectively secrete mineralized substances. Through this process, the exposed pulp area can be filled with tertiary dentin. Since MTA induces the formation of tertiary dentin, it is considered a bioactive substance. However, it is known that MTA is different from physiological dentin or reactive dentin with dentinal tubules, and induces the formation of reparative dentin similar to bone.

[0179] refer to Figure 6 It was confirmed that the absorbance of the group treated with MTA and KH001 was statistically significantly increased compared with the control group at all time points on days 7 and 14. On day 14, the degree of mineralization capacity of the group treated with MTA and KH001 showed a statistically significant difference compared with the group treated with MTA alone.

[0180] Example 3-5. Evaluation of wound healing ability and cell migration ability treated with the composition for covering dental pulp

[0181] In a 6-well plate, 1×10 5 Human dental pulp cells (passage number 2-4) were seeded at a density of 10 cells / well and then cultured in 10% α-MEM.

[0182] On the day after seeding the cells, a cell-free "wound site" was created by scraping the center of each well with a 200 μL pipette tip.

[0183] Afterwards, each photograph was analyzed using the ImageJ program after being photographed using a 10x objective lens of an upright microscope 24 hours later. All experiments consisted of three groups.

[0184] Figure 7 The purpose of this study was to confirm the efficacy of KH001 in an experiment with human dental pulp cells treated with MTA-conditioned media and to observe changes in wound healing ability and cell migration ability.

[0185] Pulp capping using MTA is applicable when odontoblasts and pulp cells are damaged. This state is a state in which there is a "wound" in the dental pulp, and a process in which surrounding cells gather to the site of cell loss or damage is required. Therefore, as a cell experimental model that mimics this process, the changes in wound healing ability and cell migration ability of the composition used for covering the dental pulp were observed using a wound healing model.

[0186] refer to Figure 7 In order to observe the effect of KH001 on the wound healing ability and cell migration ability of odontoblasts in the environment of MTA treatment, wound healing analysis was performed. Although the results were not statistically significant, the group treated with MTA showed a tendency of decreased cell migration ability compared to the control group (Ctrl). On the contrary, in the group treated with MTA and KH001 together, it was confirmed that the cell migration ability tended to recover to a level similar to that of the control group. From these results, it can be seen that in the case of pulp exposure, it is more advantageous to treat a product that combines MTA and KH001 than to use only MTA.

[0187] Example 3-6. Evaluation of the ability of the composition for covering dental pulp (MTA prototype) to penetrate into dentinal tubules

[0188] 1. Preparation of Beagle

[0189] For the in vivo test, a total of 10 beagle dogs (two years old) were used. Depending on the periodontal status of each beagle dog, 4 to 6 maxillary premolars and 6 mandibular premolars were used.

[0190] The control group and experimental group were assigned to their own beagle dogs to eliminate the possible errors caused by individual nutrition, behavior, and pulp status. All experiments using animals were performed according to the protocols approved by the Animal Care Committee of Seoul National University Animal Hospital (SNU-180416-2-1, SNU-171020-5-2).

[0191] 2. Cell Penetration Ability Assessment

[0192] According to the manufacturer's instructions, a liquid containing 10 μg / mL of CPNE7 functional peptide (KH001) and ENDOCEM MTA powder were mixed in vivo and applied to the cervical cavity close to the pulp of beagle dogs' teeth. The penetration ability of the composition for pulp covering (MTA prototype) into the dentinal tubules was then observed.

[0193] A disc-shaped Class V cavity was formed in the cervical region of a beagle dog's tooth at a depth of half the diameter of a #4 round bur, and then MTA containing a CPNE7 functional peptide (KH001) linked to fluorescent rhodamine was applied. The cavity was then fixed with 4% PFA and cut in the sagittal direction with a digital low-speed diamond cutter to prepare a sample with a thickness of 0.1 mm.

[0194] Figure 8 These are photographs taken using a confocal microscope in order to observe the ability of the composition for covering the dental pulp to penetrate into the dentinal tubules. Figure 8 A is a schematic diagram of the sample preparation process, and B and C are the results of observing whether KH001 is reached in each dentin and pulp.

[0195] refer to Figure 8 It can be seen that when 10ug / mL of CPNE7 functional peptide is directly mixed with ENDOCEM MTA powder, CPNE7 functional peptide flows smoothly along the dentinal tubules and reaches the dental pulp.

[0196] In the case of actual application of MTA, even if a much thicker dentin is left than when the pulp is exposed or a thin layer of dentin close to the pulp is left, the CPNE7 functional peptide can still reach the pulp smoothly through the dentinal tubules. Therefore, it can be expected that when performing pulp capping, the composition for covering the pulp using the CPNE7 functional peptide can seal the exposed pulp and provide an effective repair effect.

[0197] The research was conducted under the 2021 National Research and Development Project of the Ministry of Science and ICT, Welfare and Industry (KEIT) of Korea (1711138080, 1711138079; "Development of a new generation of dental restorative and bonding materials that induce tooth dentin regeneration)".

[0198] The preferred embodiments of the present invention are disclosed in this specification and the accompanying drawings. Although specific terms are used, they are used in a general sense only to easily explain the technical content of the present invention and to help understand the invention, and are not used to limit the scope of the present invention. In addition to the embodiments disclosed herein, other variations based on the technical ideas of the present invention may also be implemented, which is obvious to a person skilled in the art of the present invention.

Claims

1. A composition for bonding dentin, comprising a peptide consisting of an amino acid sequence of the following general formula 1, in, KY-R1-R2-R3-R4-R5-R6-R7-R8 (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); and R7 and R8 are lysine (K) or tyrosine (Y).

2. The composition according to claim 1, It is characterized in that The composition for bonding dentin is any one of the amino acid sequences from sequence number 1 to sequence number 96.

3. A composition for covering a dental pulp, comprising a mineral trioxide aggregate and a peptide consisting of an amino acid sequence of the following general formula 1, in, KY-R1-R2-R3-R4-R5-R6-R7-R8 (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); and R7 and R8 are lysine (K) or tyrosine (Y).

4. The composition for covering a dental pulp according to claim 3, It is characterized in that The composition for covering the dental pulp is any one of the amino acid sequences from sequence number 1 to sequence number 96.

5. A polynucleotide encoding the peptide according to claim 1 or 3.

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

7. The composition according to claim 1, It is characterized in that The composition comprises a polypeptide formed by repeatedly linking the peptides.

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

9. The composition for bonding dentin according to claim 8, It is characterized in that The damaged tooth substance is carious or fractured tooth substance.

10. The composition according to claim 1, It is characterized in that The composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

11. A method for improving or treating damaged dentin by administering the composition of claim 1 or 3 to an individual other than a human, thereby preventing necrosis of dentin, tooth removal or tooth loss after treatment.

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