Mineralizing materials and their use for treating dentin hypersensitivity and promoting epitaxial growth of enamel - Patents.com

Abstract

The present application discloses a mineralization material for treating dentin hypersensitivity and promoting epitaxial growth of enamel, and its use, which comprises, by weight, 5-72 parts of a membrane-forming protein or PEGylated membrane-forming protein, 4-67 parts of a water-soluble disulfide exchanger, and 1-21 parts of a pH adjuster. The present application first proposes using a non-toxic disulfide exchanger (e.g., cysteine ​​(Cys)) to react with a membrane-forming protein or PEGylated membrane-forming protein, so that when a protein nano-coating layer with a mineralization-regulating function is formed, a large amount of high molecular weight protein aggregates that block dentin pores are not generated, the permeability is better, and the protein nano-coating layer can penetrate deeper into the already exposed dentin pores, reaching a depth of 200-500 μm (the blocking depth of the protein coating layer reacting with TCEP is only about 40 μm), and can reach the recesses of pits and fissures.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to a Chinese patent application filed with the State Intellectual Property Office of the People's Republic of China, bearing application number 202210109256.0, filing date January 28, 2021, and entitled "Mineralizing material for treating hypersensitivity and promoting epitaxial growth of enamel and its use," which is incorporated herein by reference in its entirety. [Technical field]

[0002] This application relates to the technical field of biomaterials, and in particular to mineralizing materials and their uses for treating hypersensitivity and promoting epitaxial growth of enamel. [Background technology]

[0003] According to statistics, 15.2% to 57.4% of adults have experienced tooth sensitivity, and the incidence of dental caries in children reaches 66.9% to 88.1%, but the implementation rate of pit and fissure sealing services in children is only 22% in 2020. Oral and dental health is becoming increasingly important.

[0004] Enamel is the hardest outer surface of teeth and the first line of defense to protect dental health. Its main component is hydroxyapatite, which is difficult to demineralize in a healthy oral environment, but when various bacteria are enclosed for a long time by food residues, etc., lactic acid and other substances are generated, causing irreversible demineralization of the outermost layer of enamel, which leads to further caries and causes pit and fissure caries and dentin hypersensitivity. Pits and fissures are located in the depressions on the occlusal surface of teeth and come in various shapes, of which I-type, IK-type, and inverted Y-type account for more than 50%. Because of their special shape, they are difficult to clean thoroughly, becoming a site where plaque biofilm and cariogenic substrates stagnate, ultimately causing pit and fissure caries. Dental hypersensitivity mainly refers to the exposure of dentin due to defects in enamel, and further exposure of small holes on the dentin, and is caused by external stimuli such as hot and cold foods, sour and spicy foods, and pressure irritating the dental pulp through the small holes. Sealing of the pits is the primary means of reducing or curing sensitivity, and further restoration of the enamel prevents exposure of the dentin, providing a therapeutic effect against sensitivity and caries.

[0005] Saliva contains supersaturated calcium and phosphorus ions necessary for remineralization, and common supplements use fluoride and other substances to enhance the remineralization process to treat dentin hypersensitivity and repair caries. However, the effects of this method do not last long, and the remineralized layer is unstable and easily falls off, so dentin hypersensitivity is very likely to recur, and the effect of remineralizing enamel is unclear.

[0006] In addition, the currently proposed mineralization materials such as PTL / C-AMG or Lyso-PEG generally require the participation of a disulfide reducing agent called tris(2-carboxyethyl)phosphine hydrochloride (TCEP), which has biological safety issues, in the reaction. TCEP is harmful to the human body and can cause bad irritation to the oral tissues of the user, so its use is limited to scientific research by professionals and cannot be used in clinical diagnosis or treatment, much less in food and medicine. In addition, when lysozyme or PEGylated lysozyme reacts with TCEP, it produces high molecular weight protein aggregates, which reduces permeability. The cost is very high because the expensive amelogenin peptide (C-AMG) custom-made for the above-mentioned mineralization coating layer is also required. The already reported triethylamine-fixed ACP remineralization system also has limited clinical use due to the presence of triethylamine, which has a strong odor and is toxic.

[0007] Currently, most pit and fissure sealants are polymer resin-based, but their structure is very different from that of enamel itself, and the main drawback of conventional pit and fissure sealants is that the margins do not adhere well and the rate of fall-off is high. Therefore, the most preferred pit and fissure sealant method at present is to seal deeply and generate remineralized HAp through epitaxial growth of enamel. The two main technical problems to be solved in clinical practice at present are how to penetrate the mineralizing material deeply into the small holes or pits and fissures, and how to generate a remineralized HAp layer. Summary of the Invention [Problem to be solved by the invention]

[0008] The present application has been made to overcome the above-mentioned drawbacks, and aims to provide a mineralizing material and its use for treating hypersensitivity and promoting epitaxial growth of enamel.

[0009] This application first proposes to use a non-toxic disulfide exchanger (such as cysteine ​​(Cys)) and react it with membrane-forming proteins or PEGylated membrane-forming proteins, which when forming a protein nano-coating layer with mineralization regulation function, does not produce a large amount of high molecular weight protein aggregates that block dentin pores, has better permeability, and can penetrate deeper into the already exposed dentin pores, reaching a depth of about 200 μm (the sealing depth of the protein coating layer reacting with TCEP is only about 40 μm), and reaches the recesses of pits and fissures. The protein coating layer capable of regulating mineralization can regulate the remineralization process by attracting supersaturated calcium ions and phosphorus ions in saliva through the effects of electrostatic attraction, hydrophobic interaction, van der Waals force, etc., to generate a regenerated HAp layer to block pores and repair enamel, thereby treating caries and pit and fissure caries. The protein coating layer proposed in this application for regulating remineralization does not require special protein processing or custom-made expensive amelogenin peptides, and the protein coating layer is colorless and transparent, easy to prepare, inexpensive, non-toxic, and has excellent biocompatibility. The remineralized HAp layer has a dense structure and high stability, and reaches the inside of dentin holes and pit and fissure depressions with a mineralization depth of 200 μm. The mineralization material proposed in this application can effectively treat hypersensitivity and caries for a long period of time, and can be used to seal pits and fissures, which is of great significance in clinical use. [Means for solving the problem]

[0010] The technical solutions adopted in this application to achieve the above objectives are as follows: In a first aspect, the present application provides a mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel, comprising as ingredients, on a weight part basis, 5 to 72 parts of a membrane-forming protein or a pegylated membrane-forming protein, 4 to 67 parts of a water-soluble disulfide exchanger, and 1 to 21 parts of a pH adjuster.

[0011] Furthermore, the raw materials include, by weight, 30 to 70 parts of a membrane-forming protein or a pegylated membrane-forming protein, 20 to 67 parts of a water-soluble disulfide exchanger, and 4 to 21 parts of a pH adjuster, and optionally 30 to 50 parts of a membrane-forming protein or a pegylated membrane-forming protein, 10 to 50 parts of a water-soluble disulfide exchanger, and 6 to 20 parts of a pH adjuster.

[0012] Furthermore, the water-soluble disulfide exchange agent is a substance that has a mercapto group and is capable of carrying out a thiol-disulfide exchange reaction with a protein, and is optionally one or more of cysteine ​​and glutathione.

[0013] Furthermore, the membrane-forming protein may be any one or more of lysozyme, bovine serum albumin, human serum albumin, whey-derived albumin, insulin, α-lactalbumin, fibrinogen, β-lactoglobulin, ribonuclease A, cytochrome c, α-amylase, pepsin, myoglobin, albumin, collagen, keratin, soy protein, hemoglobin, DNA polymerase, casein, lactoferrin, trypsin, chymotrypsin, thyroglobulin, transferrin, fibrinogen, goat serum, fetal bovine serum, mouse serum, immunoglobulin, milk protein, ovalbumin, concanavalin, fish skin-derived collagen, superoxide dismutase, pancrelipase, laccase, histone, collagenase, cellulase, gluten, mucin, transglutaminase, and β-galactosidase. The PEGylated membrane forming proteins include PEGylated lysozyme, PEGylated bovine serum albumin, PEGylated human serum albumin, PEGylated whey-derived albumin, PEGylated insulin, PEGylated α-lactalbumin, PEGylated fibrinogen, PEGylated β-lactoglobulin, PEGylated ribonuclease A, PEGylated cytochrome c, PEGylated α-amylase, PEGylated pepsin, PEGylated myoglobin, PEGylated albumin, PEGylated collagen, PEGylated keratin, PEGylated soy protein, PEGylated hemoglobin, PEGylated DNA polymerase, PEGylated casein, PEGylated lactoferrin, PEGylated trypsin ... PEGylated chymotrypsin, PEGylated thyroglobulin, PEGylated transferrin, PEGylated fibrinogen, PEGylated goat serum, PEGylated fetal bovine serum, PEGylated mouse serum, PEGylated immunoglobulin, PEGylated milk proteins, PEGylated ovalbumin, PEGylated concanavalin, PEGylated fish skin collagen, PEGylated superoxide dismutase, PEGylated pancrelipase, PEGylated laccase, PEGylated histone, PEGylated collagenase, PEGylated cellulase, PEGylated gluten, PEGylated mucin, PEGylated transglutaminase, and PEGylated β-galactosidase. Optionally, the number average molecular weight of the polyethylene glycol used in the pegylated membrane-forming protein is 200 to 20,000, and is optionally lysozyme-PEG6000, lysozyme-PEG4000, or lysozyme-PEG2000, preferably lysozyme-PEG2000.

[0014] Further, the pH adjuster is selected from one or more of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium benzoate, and sodium citrate, and optionally from one or more of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate, and disodium hydrogen phosphate, and optionally from one or more of sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate, and optionally from one or two of sodium carbonate and sodium bicarbonate.

[0015] In a second aspect, there is provided a mouthwash for treating dentin hypersensitivity and promoting epitaxial growth of enamel, comprising a mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel, and optionally further comprising a buffer and additives for diluting the mineralizing material.

[0016] Furthermore, the mouthwash is obtained by uniformly mixing the mineralization material, HEPES buffer solution, and additives in a mass:volume:mass ratio of 5-25 mg:10-200 mL:5-15 mg. Optionally, in the mouthwash, the concentration of mineralizing material is greater than 0.2 mg / mL, optionally the concentration of mineralizing material is greater than 0.25 mg / mL, optionally the concentration of mineralizing material is greater than 0.3 mg / mL, optionally the concentration of mineralizing material is greater than 0.5 mg / mL, and optionally in the mouthwash, the concentration of membrane forming protein or pegylated membrane forming protein is 0.2 mg / mL or more. Optionally, the concentration range of the HEPES buffer is 5 to 20 mM, optionally 10 mM, the additives are one or more selected from ethanol, sodium citrate, sodium saccharin, 1,2-octanediol and sorbose, and optionally the pH of the mouthwash is 7 to 7.5.

[0017] In a third aspect, there is provided a dental desensitizing agent for promoting epitaxial growth of enamel, comprising a HEPES buffer, a mineralizing material for treating said hypersensitivity and promoting epitaxial growth of enamel. Optionally, the mineralizing material is prepared as a 0.1-320 mg / mL dental desensitizer solution in HEPES buffer, optionally having a concentration of 0.2-320 mg / mL, optionally having a concentration of 0.3-320 mg / mL, optionally having a concentration of 0.5-320 mg / mL, optionally having a concentration of 1-320 mg / mL, and optionally having a concentration of 0.2 mg / mL or more of the membrane forming protein or pegylated membrane forming protein in the dental desensitizer. Optionally, the concentration of the HEPES buffer is 5 to 20 mM, optionally 10 mM; optionally, the pH of the dental desensitizer solution is 7 to 7.5; and optionally, a method of using the dental desensitizer includes applying the dental desensitizer to a cotton swab and evenly applying it to dentin or using a container to immerse the dentin in the dental desensitizer solution for 1 to 5 minutes.

[0018] In a fourth aspect, there is provided a dentifrice for treating hypersensitivity and promoting epitaxial growth of enamel, comprising a dentifrice additive, and a mineralizing material for treating hypersensitivity and promoting epitaxial growth of enamel, optionally wherein the mass ratio of the mineralizing material to the dentifrice additive is 1:2-5, and optionally wherein the dentifrice additive is any one or more selected from an abrasive, a humectant, a thickener, a preservative, a colorant, and a flavoring.

[0019] In a fifth aspect, there is provided a pit and fissure sealant for treating dentin hypersensitivity and promoting epitaxial growth of enamel, comprising the mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel, the pit and fissure sealant being obtained by uniformly mixing the mineralizing material and a HEPES buffer in a mass ratio of 1:0.1-5, optionally having a concentration of 5-20 mM, optionally 10 mM, and optionally having a pH of 7-7.5 for the pit and fissure sealant solution. Effect of the Invention

[0020] (1) The present invention uses a non-toxic disulfide exchanger (e.g., cysteine ​​(Cys)) and reacts it with a membrane-forming protein or a PEGylated membrane-forming protein to form a protein nano-coating layer with a mineralization-regulating function. During the process of regulating the mineralization process, the protein nano-coating layer does not produce a large amount of high molecular weight protein aggregates, so it is possible to avoid blocking the dentin pores, and has better permeability and can enter the already exposed dentin pores to a depth of about 200 μm. Moreover, it can regulate the remineralization process by attracting supersaturated calcium ions and phosphorus ions in saliva through the effects of electrostatic attraction, hydrophobic interaction, van der Waals force, etc., so that a HAp layer with a certain degree of orientation and high stability can be formed in a short time.

[0021] (2) The mineralization material of the present application does not require special protein processing in the process of forming the protein coating layer, nor does it require custom-made expensive amelogenin peptides. The protein coating layer proposed in the present application to regulate remineralization is colorless and transparent, easy to prepare, inexpensive, non-toxic, and has excellent biocompatibility. The remineralization HAp layer has a dense structure and high stability, and can mineralize to the inside of dentin pits and fissures to a depth of 200 μm.

[0022] (3) The membrane-forming protein or PEGylated membrane-forming protein and cysteine ​​of the present application undergo a phase transition in the oral cavity, forming a two-dimensional nano-order protein film on the teeth (solid-liquid interface). The film has a large number of functional groups on its surface, so that supersaturated calcium ions and phosphate ions in saliva can be adsorbed to the surface of the film by electrostatic attraction, hydrophobic interaction, van der Waals force, etc., to form nucleation sites. The inside of the dentin hole and the surface of the missing enamel are remineralized in the saliva in the oral cavity, and a remineralized coating layer is formed, thereby deeply sealing the hole and achieving the purpose of treating hypersensitivity, repairing enamel, preventing caries, and sealing pits and fissures. As the mineralization time increases, the sealing depth for the hole reaches about 500 μm, and the thickness of the remineralized enamel also increases linearly with the mineralization time (see Figure 5). The hydroxyapatite produced by surface remineralization has a certain degree of orientation, and its performance is comparable to that of natural enamel.

[0023] (4) The mineralizing material of the present application can be used to manufacture dental desensitizers, mouthwashes, multipurpose dentifrices, pit and fissure sealants, etc. The dental desensitizers, mouthwashes, multipurpose dentifrices, and pit and fissure sealants of the present application utilize a phase transition reaction between membrane-forming protein molecules or PEGylated membrane-forming protein molecules and a water-soluble disulfide exchanger to form a protein nanofilm capable of regulating remineralization with a thickness of about 20 to 45 nm inside small holes in dentin, on the surface of missing enamel, and inside pits and fissures by induced self-organization at the interface, and the film is colorless and transparent, has good adhesion, and has the function of reinforcing hydroxyapatite newly formed by remineralization on the surface of enamel.

[0024] (5) The present application uses a protein as the main ingredient, which can effectively seal small holes to treat dentin hypersensitivity, effectively repair enamel, prevent enamel erosion and demineralization, and prevent the occurrence of caries and pit and fissure caries. The present application focuses on the sealing depth of dentin small holes, the effect of remineralization and sealing effect on the surface of enamel and inside of pits and fissures. Since dentin hypersensitivity occurs when dentin is exposed through small holes and stimulates the nerves of the teeth, caries and pit and fissure caries are first recognized as demineralization of the surface of enamel. Therefore, by sealing deep inside the dentin pits and remineralizing the enamel surface by epitaxial growth, caries can be stopped and repaired before permanent structural damage occurs in the tooth, or after permanent structural damage occurs, dentin pits can be sealed deep to treat hypersensitivity, and enamel remineralization can repair enamel defects and prevent the progression of caries or pit and fissure caries. After using the dental desensitizer, mouthwash, multi-purpose dentifrice, or pit and fissure sealant for a certain period of time, remineralization occurs on the demineralized enamel surface and inside the pits, or normal teeth are mineralized to prevent the occurrence of caries and suppress dentin hypersensitivity. The contact time of the dental desensitizer, mouthwash, multi-purpose dentifrice or pit and fissure sealant with the teeth should be about 20 seconds to about 2 minutes, and during use, a two-dimensional protein nanofilm capable of regulating remineralization is formed inside the small holes, on the surface of the enamel, and in the depressions of the pits and fissures, and then remineralization occurs in the saliva in the oral cavity inside the small holes, on the surface of the enamel, and in the depressions of the pits and fissures, forming an HAp layer.

[0025] (6) The dental desensitizer, mouthwash, multi-purpose dentifrice, or pit and fissure sealant made from the mineralization material of the present application is mainly composed of protein, and the water-soluble disulfide exchanger is also a non-toxic protein, so it meets the FDA's definition of a safe substance, is not harmful to the human body, and has excellent biocompatibility when used in Class II medical devices. In addition, the mineralization material of the present application can also be used to make other products for desensitizing teeth and preventing demineralization of enamel. [Brief description of the drawings]

[0026] One or more embodiments are illustratively described in the images shown in the drawings that correspond to them, and these illustrative descriptions are not intended to constitute limitations on the embodiments. As used herein, the word "exemplary" means "as an example, example, or for illustrative purposes." Any embodiment described herein as "exemplary" should not necessarily be construed as better or superior to other embodiments. [Figure 1] 1 shows the nanofilm and deep film formation formed on the surface of enamel and inside small holes in dentin by the mouthwash of Test Example 1 and the dental desensitizer of Test Example 2 of the present application, respectively, where a shows the atomic force of the two-dimensional nanofilm formed by biological proteins on the surface of enamel after gargling with the mouthwash corresponding to Example 1, and b shows three-dimensional imaging by a confocal laser microscope showing that the dental desensitizer corresponding to Example 1 induces deep film formation inside small holes in dentin. [Diagram 2] This is the boundary line for membrane formation under the influence of two factors, lysozyme (Lyz) concentration and cysteine ​​(Cys) concentration, in Test Example 3 of the present application. [Diagram 3]This is a scanning electron microscope image testing the newly formed remineralized coating layer on the enamel surface in a mouthwash test in Test Example 1 of the present application. Here, a is the surface of eroded enamel, b is a cross-section of the enamel, c is an energy spectrum analysis of blank enamel, d is a diagram showing the surface of the enamel after calcification was induced for two weeks in the improved artificial saliva using a mouthwash corresponding to Example 1, e is a diagram showing a cross-section of the enamel after calcification was induced for two weeks in the improved artificial saliva using a mouthwash corresponding to Example 1, f is a diagram showing an energy spectrum analysis of the surface of the enamel after calcification was induced for two weeks in the improved artificial saliva using a mouthwash corresponding to Example 1, g is a diagram showing the surface of enamel after calcification was induced for three days in actual saliva in the oral cavity using a mouthwash corresponding to Example 1, h is a diagram showing a cross-section of enamel after calcification was induced for three days in actual saliva in the oral cavity using a mouthwash corresponding to Example 1, and i is a diagram showing an energy spectrum analysis of the surface of enamel after calcification was induced for three days in actual saliva in the oral cavity using a mouthwash corresponding to Example 1. [Figure 4] 1 shows scanning electron microscope images of blank dentin sections and dentin sections coated with a dental desensitizer corresponding to Example 1, where a is the surface of blank dentin, b is the surface of dentin coated with a dental desensitizer corresponding to Example 1, c is a cross section of blank dentin, d is a cross section of dentin after 7 days of biomineralization without dental desensitizer application, e is a cross section of dentin after 7 days of biomineralization with dental desensitizer application corresponding to Example 1 (maximum depth reaches about 200 μm), and f is an energy spectrum analysis of remineralization crystals after dental desensitizer application corresponding to Example 1. [Diagram 5]Scanning electron microscope images of enamel using mouthwashes corresponding to Comparative Example 1 and Example 1, and the change in mineralization thickness over mineralization time, where a is a diagram showing the surface of enamel after using the mouthwash of Comparative Example 1 for two weeks, b is a diagram showing a cross-section of remineralized enamel after using the mouthwash of Comparative Example 1 for two weeks, c is a diagram showing the surface of enamel after using the mouthwash corresponding to Example 1 of the present application for two weeks, d is a diagram showing a cross-section of remineralized enamel after using the mouthwash corresponding to Example 1 of the present application for two weeks, and e is the change in thickness of the mineralized material over mineralization time when using the mouthwash corresponding to Example 1 of the present application. [Figure 6] 1 shows scanning electron microscope images of dentin using dental desensitizers corresponding to Comparative Example 2 and Example 1. Here, a is a diagram showing the surface of dentin after using the desensitizer obtained in Comparative Example 2 for 3 weeks, b is a cross-section of remineralized dentin after using the desensitizer obtained in Comparative Example 2 for 3 weeks, c is a diagram showing the surface of dentin after using the dental desensitizer corresponding to Example 1 of the present application for 3 weeks, and d is a diagram showing the cross-section of remineralized dentin after using the dental desensitizer corresponding to Example 1 of the present application for 3 weeks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] In the following, the technical solutions of the embodiments of the present application are clearly and completely described, so as to make the objectives, technical solutions and advantages of the embodiments of the present application clearer. Needless to say, the described embodiments are only some of the embodiments of the present application, and are not all of the embodiments. All other embodiments that a person skilled in the art can obtain based on the embodiments of the present application without inventive work shall fall within the scope of protection of the present application.

[0028] In addition, in order to further explain the present application, many specific details are shown in the following specific embodiments. It is understood by those skilled in the art that the present application can be similarly implemented without some of these specific details. In some examples, in order to enhance the gist of the present application, materials, elements, methods, means, etc. familiar to those skilled in the art are not described in detail.

[0029] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations thereof such as "including" or "containing" are to be understood to include the described elements or components and not to exclude other elements or components.

[0030] Example 1 70 mg of lysozyme, 65 mg of cysteine ​​(Cys), and 20 mg of sodium bicarbonate were homogeneously mixed to obtain a mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel.

[0031] Example 2 30 mg of lysozyme, 46 mg of cysteine ​​(Cys), and 15.2 mg of sodium bicarbonate were mixed uniformly to obtain a mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel.

[0032] Example 3 40 mg of lysozyme, 20.8 mg of glutathione (GSH), and 6.2 mg of sodium bicarbonate were homogeneously mixed to obtain a mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel.

[0033] Example 4 52 mg of lysozyme, 65 mg of glutathione (GSH), and 18.2 mg of sodium bicarbonate were homogeneously mixed to obtain a mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel.

[0034] Example 5 72 mg of PEGylated lysozyme, 67 mg of cysteine ​​(Cys), and 20.6 mg of sodium bicarbonate were mixed uniformly to obtain a mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel.

[0035] Example 6 62 mg of PEGylated lysozyme, 50 mg of glutathione (GSH), and 16.2 mg of sodium bicarbonate were homogeneously mixed to obtain a mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel.

[0036] Example 7 The mouthwash of this example consists of each of the mineralized materials obtained in Examples 1 to 6 above and 10 mM HEPES buffer.At the time of use, 6.2 mg of mineralized material was placed in 40 mL of 10 mM HEPES buffer, 5 mg of sodium saccharin and 2 mg of sorbose were added and mixed uniformly to obtain a mouthwash for treating hypersensitivity and promoting epitaxial growth of enamel.

[0037] Example 8 The mouthwash of this example consists of each of the mineralized materials obtained in Examples 1 to 6 above and 10 mM HEPES buffer.At the time of use, 20 mg of the mineralized material was placed in 240 mL of 10 mM HEPES buffer, 6 mg of sodium saccharin and 4 mg of sorbose were added and mixed uniformly to obtain a mouthwash for treating hypersensitivity and promoting epitaxial growth of enamel.

[0038] Example 9 The dental desensitizer of this embodiment consists of each of the mineralized materials obtained in Examples 1 to 6 above and 10 mM HEPES buffer solution. At the time of use, 35 mg of the mineralized material and 120 mL of 10 mM HEPES buffer solution were uniformly mixed to obtain a dental desensitizer for treating hypersensitivity and promoting epitaxial growth of enamel.

[0039] Example 10 The dental desensitizer of this example consists of each of the mineralized materials obtained in Examples 1 to 6 above and 10 mM HEPES buffer solution, and at the time of use, 52 mg of the mineralized material and 152 mL of 10 mM HEPES buffer solution were uniformly mixed to obtain a dental desensitizer for treating hypersensitivity and promoting epitaxial growth of enamel.

[0040] Example 11 The pit and fissure sealant of this example consisted of each of the mineralized materials obtained in Examples 1 to 6 above and 10 mM HEPES buffer solution. At the time of use, 42 mg of the mineralized material was uniformly mixed with 25 mL of 10 mM HEPES buffer solution to obtain a pit and fissure sealant for treating dentin hypersensitivity and promoting epitaxial growth of enamel.

[0041] Example 12 The pit and fissure sealant of this example consisted of each of the mineralized materials obtained in Examples 1 to 6 above and 10 mM HEPES buffer solution. At the time of use, 26.2 mg of the mineralized material was uniformly mixed with 30.2 mL of 10 mM HEPES buffer solution to obtain a pit and fissure sealant for treating dentin hypersensitivity and promoting epitaxial growth of enamel.

[0042] (Example 13) The pit and fissure sealant of this embodiment is composed of the mineralized material obtained in Examples 1 to 6 above and a dentifrice additive. At the time of use, 12 mg of the mineralized material and 20 mg of the dentifrice additive were uniformly mixed to obtain a multi-purpose dentifrice for treating hypersensitivity and promoting epitaxial growth of enamel.

[0043] Example 14 The pit and fissure sealant of this embodiment is composed of the mineralized material obtained in Examples 1 to 6 above and a dentifrice additive. When used, 45 mg of the mineralized material and 100 mg of the dentifrice additive are uniformly mixed to obtain a multi-purpose dentifrice for treating hypersensitivity and promoting epitaxial growth of enamel.

[0044] In the above examples, lysozyme or PEGylated lysozyme may be selected from the group consisting of bovine serum albumin, human serum albumin, whey-derived albumin, insulin, α-lactalbumin, fibrinogen, β-lactoglobulin, ribonuclease A, cytochrome c, α-amylase, pepsin, myoglobin, albumin, collagen, keratin, soy protein, hemoglobin, DNA polymerase, casein, lactoferrin, trypsin, chymotrypsin, thyroglobulin, transferrin, fibrinogen, yasum, and the like. The PEGylated membrane forming protein may be replaced by any one or more of the following: goat serum, fetal bovine serum, mouse serum, immunoglobulins, milk proteins, ovalbumin, concanavalin, collagen from fish skin, superoxide dismutase, pancrelipase, laccase, histones, collagenase, cellulase, gluten, mucin, transglutaminase, β-galactosidase, or the PEGylated membrane forming protein may be replaced by any one or more of the following: PEGylated lysozyme, PEGylated bovine serum albumin, PEGylated human serum albumin, PEGylated whey-derived albumin, ... lysozyme, PEGylated bovine serum albumin, PEGylated human serum albumin, PEGylated lysozyme, PEGylated bovine serum albumin, PEGylated human serum albumin, PEGylated lysozyme, PEGylated bovine serum albumin, PEGylated human serum albumin, PEGylated lysozyme, PEGylated bovine serum albumin, PEGylated human serum albumin, pegylated insulin, pegylated alpha-lactalbumin, pegylated fibrinogen, pegylated beta-lactoglobulin, pegylated ribonuclease A, pegylated cytochrome c, pegylated alpha-amylase, pegylated pepsin, pegylated myoglobin, pegylated albumin, pegylated collagen, pegylated keratin, pegylated soy protein, pegylated hemoglobin, pegylated DNA polymerase, pegylated casein, pegylated lactoferrin, pegylated trypsin, pegylated chymotrypsin, pegylated thyroglobulin, pegylated transferrin, The present invention relates to a method for the preparation of collagenase from fish skin, comprising administering to the patient a therapeutically effective amount of a PEG-1000 stimulant ...

[0045] Comparative Example 1 20 mg of lysozyme, 10 mg of amelogenin peptide, 10 mg of disodium hydrogen phosphate, 5.2 mg of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and 3.5 mg of sodium dihydrogen phosphate were uniformly mixed to obtain the calcified material of this comparative example.

[0046] 2 mg of the mineralizing material of this comparative example was added to 10 mL of HEPES buffer, and 8 mg of sodium saccharin and 2 mg of sorbose were further added and mixed uniformly to obtain a mouthwash for preventing enamel demineralization of this comparative example. The concentration of the HEPES buffer in this comparative example was 20 mM.

[0047] Comparative Example 2 40 mg of PEGylated lysozyme, 10 mg of calcium chloride, 10 mg of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and 60 mg of sodium hydrogen carbonate were uniformly mixed to obtain the desensitizing agent of this comparative example.

[0048] 60 mg of the mineralized material of this comparative example was added to 10 mL of deionized water and mixed uniformly to obtain a 6 mg / mL desensitizer solution of this comparative example.

[0049] In order to prove the beneficial effects of the present application, the inventor conducted a performance test of the mouthwash obtained in Example 8 for treating hypersensitivity and promoting epitaxial growth of enamel, and the test was specifically as follows: (Test Example 1) Human third molar samples without caries were collected (tooth samples were provided by Stomatology Hospital of Air Force Military Medical University and Stomatology Hospital of Tianjin Medical University, and the study was approved by the medical research ethics committee of the vendor). After cleaning, the enamel was processed with a SYJ160 low-speed microtome and 200-6000 mesh sandpaper to make enamel slices of 6mm x 6mm x 1.5mm. The polished enamel slices were etched with 37% (wt%) phosphoric acid for 30 seconds, rinsed with ultrapure water, subjected to ultrasonic waves for 15 minutes, dried by blowing nitrogen, and then placed in the mouthwash obtained in Example 8 and immersed for 1 minute to simulate the process of gargling.

[0050] After 1 minute, a two-dimensional nanofilm made of biological proteins was formed on the surface of the enamel, and the test results for the mouthwash in Example 8 (a mouthwash produced by the method of Example 8 using the mineralization material of Example 1, hereinafter referred to as the mouthwash corresponding to Example 1) showed a thickness of approximately 30 to 50 nm, as shown in Figure 1a. The mineralization conditions in the improved artificial saliva and actual saliva in the oral cavity were confirmed, respectively. 1) The enamel covered with the protein film was immersed in the modified artificial saliva and biomineralized at 37°C, and the modified artificial saliva was replaced once a day. During the mineralization process, the enamel was removed from the modified artificial saliva every day, rinsed with ultrapure water, dried by nitrogen blowing, and then immersed in the mouthwash obtained in Example 8 for 1 minute, and then transferred to the modified artificial saliva, and the process was repeated twice a day. After two weeks, the planar morphology of the remineralized coating layer of the enamel was observed with a scanning electron microscope, the remineralized coating layer was surface-scanned with an energy spectrometer to analyze the elements of the remineralized coating layer, the enamel was indented with a Knoop hardness tester, and the cross-sectional morphology of the remineralized coating layer of the enamel was observed with a scanning electron microscope, where the scanning results of the energy spectrometer when using the mouthwash corresponding to Example 1 are shown in Fig. 3d and f, and the scanning results of the Knoop hardness tester are shown in Fig. 3e.

[0051] As shown in the results in Figure 3, a two-dimensional nanofilm made of biological proteins was grown on enamel by an in situ growth method. When the enamel coated with the protein film was placed in an improved artificial saliva to simulate biomineralization, well-oriented hydroxyapatite was produced, and the calcium / phosphorus ratio was consistent with that of natural enamel. The newly formed hydroxyapatite by remineralization on the surface of eroded enamel (Figure 3 a-c) prevents enamel demineralization and prevents dental caries.

[0052] 2) The enamel on which the protein film has grown was immersed in saliva in the actual oral cavity after centrifugation (wherein the actual saliva was collected from healthy adult subjects, the samples were collected at least 2 hours after a meal, and the collection of the samples was permitted by the Medical Research Ethics Committee), and biomineralization was simulated by immersing the enamel on which the film has grown in the saliva in the actual oral cavity. After 3 days, the morphology of the enamel surface was observed, and the results are shown in Figure 3 g to i. As is clear from the figure, when the enamel on which the protein film has grown was immersed in the saliva in the actual oral cavity, regenerated hydroxyapatite with a high degree of orientation was obtained by remineralization, which proved that the protein film can exert a mineralization effect even in the saliva environment in the oral cavity and can be used in the oral cavity environment of the human body.

[0053] The inventor further compared the scanning electron microscope images of the film formation and mineralized enamel by the mouthwash of Comparative Example 1 and the mouthwash corresponding to Example 1 by the above method, and the results are shown in Figure 5. As shown in the results, when the mouthwash corresponding to Example 1 was used, the formed mineralized layer had a dense surface (Figure 5c), and the thickness of the remineralized enamel layer increased linearly with the mineralization time (Figure 5e), whereas the mouthwash of Comparative Example 1 had a non-dense surface of the mineralized enamel layer (Figure 5a), and the thickness of the remineralized enamel layer by the mouthwash of Comparative Example 1 (Figure 5b, the position indicated by the arrow) was far below the depth of the mineralization by the mouthwash corresponding to Example 1 (Figure 5d, the position indicated by the arrow).

[0054] (Test Example 2) In order to prove the beneficial effects of the present invention, the inventor further carried out a performance test of the dental desensitizer obtained in Example 10, and the test results were specifically as follows: Human third molar samples without caries were collected (tooth samples were provided by the Stomatological Hospital of Air Force Military Medical University and the Stomatological Hospital of Tianjin Medical University, and the study was approved by the medical research ethics committee of the respective vendors), cleaned, and then 2 mm thick dentin slices were prepared using a SYJ160 low-speed microtome and polished to obtain dentin samples of 5 mm × 5 mm × 2 mm. The dentin samples were cariously treated with 17% (wt%) EDTA solution and 2% (wt%) NaClO solution to be used as test samples.

[0055] The sample was immersed in the tooth desensitizer solution, left to stand at 37°C for 2 minutes, and then removed. Due to the characteristic that the tooth desensitizer can specifically bind to the fluorescent dye thioflavin T (ThT), the three-dimensional imaging test using a confocal laser microscope proved that the tooth desensitizer can penetrate into the small hole and form a film, and the test result of the tooth desensitizer in Example 10 (the tooth desensitizer manufactured by the method of Example 10 using the mineralized material of Example 1, hereinafter referred to as the tooth desensitizer corresponding to Example 1) is shown in Figure 1b.

[0056] The dentin slices coated with the dental desensitizer were left in a 12-well plate, and the improved artificial saliva was added and cultured at 37°C. The improved artificial saliva was replaced every day, and after remineralization for 7 days, the state of the small holes being sealed was observed using a scanning electron microscope. A blank slice not coated with the dental desensitizer was used as a control test, and the test results of the dental desensitizer corresponding to Example 1 are shown in Figure 4.

[0057] As can be seen from FIG. 4e, the inside of the small holes in the dentin slices on which a protein film was formed using the dental desensitizer was sealed by remineralization crystals, but no crystals appeared inside the small holes in the dentin slices of the blank control group (FIG. 4d) where no dental desensitizer was applied, and the dentin remained exposed. Energy spectrum analysis (FIG. 4f) proved that the remineralization crystals were hydroxyapatite HAp. This test example showed that the dental desensitizer proposed in this application can penetrate deep into the small holes to form a protein nano-coating layer with remineralization regulating function, and can induce remineralization of dentin in an improved artificial saliva environment, deeply sealing the small holes in dentin, thereby treating the symptoms of hypersensitivity.

[0058] The inventors further extended the remineralization time (3 weeks) using the above method, and compared scanning electron microscope images of mineralized dentin produced by the dental desensitizer of Comparative Example 2 and the dental desensitizer corresponding to Example 1, and the results are shown in Figure 6. As shown in the results, as the mineralization time increased, the dental desensitizer corresponding to Example 1 sealed the small holes to a depth of 200 to 500 μm (the distance from the surface of the dentin slice in the box in Figure 6d was about 420 μm), whereas the dental desensitizer of Comparative Example 2 sealed the small holes to a depth of only about 40 μm (indicated by the white arrow in Figure 6b).

[0059] (Test Example 3) In order to clarify the boundary line between membrane-forming protein and cysteine, the inventors prepared solutions of membrane-forming protein and cysteine ​​with different concentrations and plotted the curves in Figure 2. As can be seen from this figure, at the Lyz and Cys concentrations below the left side of the boundary line, complete two-dimensional nanofilms that can regulate the remineralization process cannot be aggregated and formed, and as can be seen from this figure, when the protein concentration is 0.2 mg / mL or higher, a small amount of cysteine ​​can form a membrane, but when the protein concentration is lower than 0.2 mg / mL, a high concentration of cysteine ​​is required for membrane formation.

[0060] As can be seen from the above, the mineralization material used in the present application is mainly protein, etc., the material is non-toxic, has excellent biocompatibility, is easy to prepare, and is easy to use, and in vitro tests have demonstrated that the crystals generated by remineralization have a stable structure and are comparable to natural enamel or dentin, enabling long-term desensitization of teeth and excellent therapeutic effects. The mineralization material used in the present application for treating hypersensitivity, repairing enamel defects, and preventing caries, as well as products such as dental desensitizers, mouthwashes, and dentifrices made from this mineralization material, are expected to have excellent therapeutic applications.

[0061] The present application uses protein lysozyme (or PEGylated lysozyme) to form a protein nanofilm through the action of a disulfide exchanger (cysteine ​​or glutathione), which can be used for the remineralization of dentin and the treatment of caries and the sealing of pits and fissures by promoting epitaxial growth of enamel. The mineralization material of the present application can enter the inside of the already exposed dentin pits, the surface of the carious enamel, and the inside of the pits and fissures by simple methods such as immersion and coating, and form a single protein nanofilm coating layer with remineralization induction function, which can induce the saturated calcium ions and phosphorus ions in saliva to remineralize the inside of the dentin pits to form a HAp layer, which can promote the remineralization of HAp on the surface of the enamel and realize the epitaxial growth of enamel, thereby achieving the purpose of treating hypersensitivity, preventing caries, and sealing of pits and fissures. In this application, the protein coating layer formed inside the dentin and on the surface of the enamel also has antibacterial and anti-fouling capabilities, and plays an important role in keeping the oral cavity clean and healthy. All of the mineralization materials mentioned in this application are safe substances certified by the U.S. Food and Drug Administration (FDA) and have better biocompatibility, so they are expected to be used more widely in clinical practice.

[0062] Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present application, and are not intended to be limiting. Although the present application has been described in detail with reference to the above embodiments, it is still understood by those skilled in the art that the technical solutions described in the above embodiments can be modified or some of the technical features thereof can be replaced with equivalents, and such modifications or replacements do not deviate from the essence and scope of the technical solutions of the embodiments of the present application. [Industrial Applicability]

[0063] The present application provides a mineralization material for treating dentin hypersensitivity and promoting epitaxial growth of enamel, comprising, by weight, 5-72 parts of a membrane-forming protein or PEGylated membrane-forming protein, 4-67 parts of a water-soluble disulfide exchanger, and 1-21 parts of a pH adjuster, and its use. The dental desensitizer, mouthwash, multi-purpose dentifrice, or pit and fissure sealant produced from the mineralization material of the present application is mainly composed of protein, and the water-soluble disulfide exchanger is also a non-toxic protein, so it is not harmful to the human body and has excellent biocompatibility. In addition, the mineralization material of the present application can also be used to produce other products for desensitizing teeth and preventing demineralization of enamel.

Claims

1. A mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel, comprising, as ingredients, on a weight part basis, 5 to 72 parts of a membrane-forming protein or a pegylated membrane-forming protein, 4 to 67 parts of a water-soluble disulfide exchanger, and 1 to 21 parts of a pH adjuster.

2. 2. The mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel according to claim 1, characterized in that it comprises, as ingredients, on a weight part basis, 30 to 70 parts of a membrane-forming protein or a pegylated membrane-forming protein, 20 to 67 parts of a water-soluble disulfide exchanger, and 4 to 21 parts of a pH adjuster, or optionally 30 to 50 parts of a membrane-forming protein or a pegylated membrane-forming protein, 10 to 50 parts of a water-soluble disulfide exchanger, and 6 to 20 parts of a pH adjuster.

3. The mineralization material for treating dentin hypersensitivity and promoting epitaxial growth of enamel as described in claim 1, characterized in that the water-soluble disulfide exchange agent is a substance having a mercapto group and capable of undergoing a thiol-disulfide exchange reaction with a protein, and is optionally one or more of cysteine ​​and glutathione.

4. As the membrane-forming protein, any one or more of lysozyme, bovine serum albumin, human serum albumin, whey-derived albumin, insulin, α-lactalbumin, fibrinogen, β-lactoglobulin, ribonuclease A, cytochrome c, α-amylase, pepsin, myoglobin, albumin, collagen, keratin, soy protein, hemoglobin, DNA polymerase, casein, lactoferrin, trypsin, chymotrypsin, thyroglobulin, transferrin, fibrinogen, goat serum, fetal bovine serum, mouse serum, immunoglobulin, milk protein, ovalbumin, concanavalin, fish skin-derived collagen, superoxide dismutase, pancrelipase, laccase, histone, collagenase, cellulase, gluten, mucin, transglutaminase, and β-galactosidase are used; The PEGylated membrane forming protein is selected from the group consisting of PEGylated lysozyme, PEGylated bovine serum albumin, PEGylated human serum albumin, PEGylated whey-derived albumin, PEGylated insulin, PEGylated α-lactalbumin, PEGylated fibrinogen, PEGylated β-lactoglobulin, PEGylated ribonuclease A, PEGylated cytochrome c, PEGylated α-amylase, PEGylated pepsin, PEGylated myoglobin, PEGylated albumin, PEGylated collagen, PEGylated keratin, PEGylated soy protein, PEGylated hemoglobin, PEGylated DNA polymerase, PEGylated casein, PEGylated lactoferrin, PEGylated trypsin ... PEGylated chymotrypsin, PEGylated thyroglobulin, PEGylated transferrin, PEGylated fibrinogen, PEGylated goat serum, PEGylated fetal bovine serum, PEGylated mouse serum, PEGylated immunoglobulin, PEGylated milk proteins, PEGylated ovalbumin, PEGylated concanavalin, PEGylated fish skin collagen, PEGylated superoxide dismutase, PEGylated pancrelipase, PEGylated laccase, PEGylated histone, PEGylated collagenase, PEGylated cellulase, PEGylated gluten, PEGylated mucin, PEGylated transglutaminase, PEGylated β-galactosidase; 4. The mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel according to any one of claims 1 to 3, characterized in that optionally, the number average molecular weight of the polyethylene glycol used in the pegylated membrane-forming protein is 200-20,000, optionally lysozyme-PEG6000, lysozyme-PEG4000, lysozyme-PEG2000, preferably lysozyme-PEG2000.

5. 2. The mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel according to claim 1, characterized in that the pH adjuster is any one or more selected from sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium benzoate and sodium citrate, optionally any one or more selected from sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate and disodium hydrogen phosphate, optionally any one or more selected from sodium carbonate, sodium bicarbonate, potassium carbonate and potassium bicarbonate, and optionally one or two selected from sodium carbonate and sodium bicarbonate.

6. A mouthwash for treating dentin hypersensitivity and promoting epitaxial growth of enamel, comprising a mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel as described in any one of claims 1 to 5, and optionally further comprising a buffer and additives for diluting the mineralizing material.

7. The mouthwash is obtained by uniformly mixing the mineralization material, the HEPES buffer, and the additive in a mass:volume:mass ratio of 5-25 mg:10-200 mL:5-15 mg; Optionally, in the mouthrinse, the concentration of mineralizing material is greater than 0.2 mg / mL, optionally the concentration of mineralizing material is greater than 0.25 mg / mL, optionally the concentration of mineralizing material is greater than 0.3 mg / mL, optionally the concentration of mineralizing material is greater than 0.5 mg / mL, optionally in the mouthrinse, the concentration of membrane forming protein or pegylated membrane forming protein is equal to or greater than 0.2 mg / mL; A mouthwash for treating hypersensitivity and promoting epitaxial growth of enamel as described in claim 6, characterized in that optionally, the concentration range of the HEPES buffer is 5 to 20 mM, optionally 10 mM, the additives are one or more selected from ethanol, sodium citrate, sodium saccharin, 1,2-octanediol and sorbose, and optionally, the pH of the mouthwash is 7 to 7.

5.

8. 1. A dental desensitizing agent for promoting epitaxial growth of enamel, comprising: HEPES buffer, a mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel according to any one of claims 1 to 5, Optionally, the mineralized material is prepared in a HEPES buffer as a 0.1-320 mg / mL dental desensitizer solution, optionally at a concentration of 0.2-320 mg / mL, optionally at a concentration of 0.3-320 mg / mL, optionally at a concentration of 0.5-320 mg / mL, optionally at a concentration of 1-320 mg / mL, and optionally in the dental desensitizer, the concentration of the membrane forming protein or pegylated membrane forming protein is 0.2 mg / mL or more; A dental desensitizer, optionally characterized in that the concentration of the HEPES buffer is 5 to 20 mM, optionally 10 mM, optionally the pH of the dental desensitizer solution is 7 to 7.5, and optionally a method of using the dental desensitizer includes applying the dental desensitizer to a cotton swab and evenly applying it to dentin or using a container to immerse dentin in the dental desensitizer solution for 1 to 5 minutes.

9. A dentifrice for treating hypersensitivity and promoting epitaxial growth of enamel, comprising a dentifrice additive, a mineralizing material for treating hypersensitivity and promoting epitaxial growth of enamel according to any one of claims 1 to 5, optionally a mass ratio of the mineralizing material to the dentifrice additive is 1:2 to 5, and optionally the dentifrice additive is one or more selected from an abrasive, a humectant, a thickener, a preservative, a colorant, and a flavoring.

10. A pit and fissure sealant for treating dentin hypersensitivity and promoting epitaxial growth of enamel, comprising the mineralizing material for treating dentin hypersensitivity and promoting epitaxial growth of enamel according to any one of claims 1 to 5, characterized in that the pit and fissure sealant is obtained by uniformly mixing the mineralizing material and a HEPES buffer solution in a mass ratio of 1:0.1 to 5, optionally having a concentration of 5 to 20 mM, optionally 10 mM, and optionally having a pH of 7 to 7.5.

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