Calcification materials and their use for treating hypersensitivity and promoting enamel epitaxial growth

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 the priority of a Chinese patent application with an application number of 202210109256.0, a filing date of January 28, 2021, and an application title of "Calcium - containing material for treating hypersensitivity and promoting epitaxial growth of enamel and its use", which was filed with the China National Intellectual Property Administration, and the said application is incorporated herein by reference in its entirety.

Technical field

[0002] This application relates to the technical field of biomaterials, and in particular, to a calcium - containing material for treating hypersensitivity and promoting epitaxial growth of enamel and its use.

Background art

[0003] According to statistics, 15.2 - 57.4% of adults have experienced being troubled by hypersensitivity, and the incidence rate of dental caries in children reaches 66.9 - 88.1%. However, the implementation rate of the service of pit - and - fissure sealant in children in 2020 is only 22%. Oral and dental health is becoming increasingly important.

[0004] Enamel is the hardest outer surface of the tooth and the first line of defense protecting dental health. Its main component is hydroxyapatite, which is resistant to demineralization in a healthy oral environment. However, when various bacteria are trapped for extended periods by food residue, lactic acid and other substances are produced, causing irreversible demineralization of the outermost layer of enamel, leading to caries, pit and fissure caries, and tooth sensitivity. Pits and fissures are located in the depressions on the occlusal surface of the tooth and come in various forms, with I-shaped, IK-shaped, and inverted Y-shaped types accounting for more than 50%. Due to their unique morphology, thorough cleaning is difficult, making them areas where plaque biofilm and cariogenic matrix accumulate, ultimately leading to pit and fissure caries. Tooth sensitivity mainly refers to the exposure of dentin due to enamel defects, and further exposure of small holes in the dentin. External stimuli such as hot or cold foods, sour or spicy foods, and pressure stimulate the pulp through these small holes. Sealing small holes is a primary means of reducing or healing hypersensitivity, and further repairing the enamel can prevent dentin exposure, thus achieving therapeutic effects against hypersensitivity and caries.

[0005] Saliva contains supersaturated calcium and phosphorus ions necessary for remineralization, and common supplements use fluoride and other ingredients to enhance the remineralization process, thereby treating hypersensitivity and repairing caries. However, this method does not provide long-lasting effects, and the remineralized layer is unstable and easily shed, making hypersensitivity very prone to recurrence, and the effectiveness of enamel remineralization is not clear.

[0006] Furthermore, currently proposed calcification materials such as PTL / C-AMG or Lyso-PEG generally require the participation of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), a disulfide reducing agent with biological safety issues, in the reaction. TCEP causes adverse irritation to the user's oral tissues and is harmful to the human body; therefore, its use is limited to scientific research by specialized companies and cannot be used for clinical diagnosis or treatment, much less in food or pharmaceuticals. In addition, lysozyme or pegylated lysozyme reacts with TCEP to form high-molecular-weight protein aggregates, reducing its permeability. The above-mentioned calcification coating also requires expensive, custom-made amelogenin peptide (C-AMG), making it very costly. 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, polymer resin-based pit and fissure sealants are the most common. However, their structure differs significantly from that of enamel itself, resulting in poor marginal adhesion and a high rate of shedding, which are considered the main drawbacks of conventional pit and fissure sealants. Therefore, deeply sealing the pit and fissure to induce remineralized HAp through epitaxial growth of enamel is currently the most preferred method for sealing pits and fissures. The two main technical challenges that need to be addressed clinically are how to deeply penetrate the pit or fissure with the calcifying material, and how to generate a remineralized HAp layer. [Overview of the project] [Problems that the invention aims to solve]

[0008] This application aims to overcome the aforementioned shortcomings and to provide a calcification material and its use for treating hypersensitivity and promoting epitaxial growth of enamel.

[0009] This invention is the first to propose using a non-toxic disulfide exchanger (e.g., cysteine ​​(Cys)) and reacting it with a film-forming protein or pegylated film-forming protein. When a protein nanocoating layer with calcification-regulating function is formed in this way, large amounts of high molecular weight protein aggregates that would otherwise block dentin pores are not generated, resulting in better permeability and the ability to penetrate deeper into the already exposed dentin pores, reaching a depth of approximately 200 μm (compared to only about 40 μm in the sealing depth of the protein coating layer reacting with TCEP), and reaching the depressions of pits and fissures. This calcification-regulating protein coating layer can treat caries and pit and fissure caries by attracting supersaturated calcium and phosphate ions in saliva through the action of electrostatic attraction, hydrophobic interactions, van der Waals forces, etc., thereby regulating the remineralization process, generating a regenerated HAp layer, blocking pores, and repairing enamel. The protein coating layer proposed in this application, which regulates remineralization, does not require special protein treatment or the custom fabrication of expensive amelogenin peptides. This protein coating layer is colorless and transparent, easy to prepare, inexpensive, non-toxic, and possesses excellent biocompatibility. The remineralizing HAp layer has a dense structure, high stability, and reaches a calcification depth of 200 μm into the interior of dentin pores and fissure depressions. The calcification material of this application can effectively treat hypersensitivity and caries over the long term and can be used for pit and fissure sealing, thus having great clinical significance. [Means for solving the problem]

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

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

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

[0013] Furthermore, as the membrane-forming protein, one or more of the following can be used: 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 aforementioned 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, and pegy It contains one or more of the following: chymotrypsin, pegylated thyroglobulin, pegylated transferrin, pegylated fibrinogen, pegylated goat serum, pegylated fetal bovine serum, pegylated mouse serum, pegylated immunoglobulin, pegylated milk protein, 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 polyethylene glycol used in the PEGylated film-forming protein is 200 to 20000, and optionally lysozyme-PEG6000, lysozyme-PEG4000, or lysozyme-PEG2000, preferably lysozyme-PEG2000.

[0014] Furthermore, the pH adjusting agent may be one or more selected from sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium benzoate, and sodium citrate, and optionally one or more selected from sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, dipotassium hydrogen phosphate, and disodium hydrogen phosphate, and optionally 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.

[0015] In a second embodiment, a mouthwash for treating hypersensitivity and promoting enamel epitaxial growth is provided, comprising a calcifying material for treating the aforementioned hypersensitivity and promoting enamel epitaxial growth, and optionally further comprising a buffer and additives for diluting the calcifying material.

[0016] Furthermore, the mouthwash is obtained by uniformly mixing the calcifying material, HEPES buffer, 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 calcifying material is greater than 0.2 mg / mL, optionally, the concentration of calcifying material is greater than 0.25 mg / mL, optionally, the concentration of calcifying material is greater than 0.3 mg / mL, optionally, the concentration of calcifying 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 higher. Optionally, the concentration range of the HEPES buffer is 5 to 20 mM, and optionally 10 mM; the additive is selected from one or more of 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, a dental desensitizer for promoting enamel epitaxial growth is provided, comprising a HEPES buffer and a calcifying material for treating the aforementioned hypersensitivity and promoting enamel epitaxial growth. Optionally, the calcification material is prepared as a dental desensitizer solution of 0.1 to 320 mg / mL using HEPES buffer, optionally with a concentration of 0.2 to 320 mg / mL, optionally with a concentration of 0.3 to 320 mg / mL, optionally with a concentration of 0.5 to 320 mg / mL, optionally with a concentration of 1 to 320 mg / mL, and optionally, in the dental desensitizer, the concentration of film-forming protein or pegylated film-forming protein is 0.2 mg / mL or higher. Optionally, the concentration of the HEPES buffer is 5-20 mM, optionally 10 mM, optionally the pH of the dental desensitizer solution is 7-7.5, and optionally the method of using the dental desensitizer includes applying the dental desensitizer uniformly to the dentin using a cotton swab or immersing the dentin in the dental desensitizer solution for 1-5 minutes using a container.

[0018] In a fourth embodiment, a toothpaste is provided for treating hypersensitivity and promoting enamel epitaxial growth, comprising a toothpaste additive and a calcifying material for treating the aforementioned hypersensitivity and promoting enamel epitaxial growth, wherein optionally the mass ratio of the calcifying material to the toothpaste additive is 1:2 to 5, and optionally the toothpaste additive is selected from one or more of abrasives, humectants, thickeners, preservatives, pigments, and fragrances.

[0019] In a fifth aspect, a pit and fissure sealing material for treating hypersensitivity and promoting epitaxial growth of enamel is provided, comprising a calcifying material for treating the aforementioned hypersensitivity and promoting epitaxial growth of enamel, wherein the pit and fissure sealing material is obtained by uniformly mixing the calcifying material and HEPES buffer 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 of the pit and fissure sealing material solution. [Effects of the Invention]

[0020] (1) This invention uses a non-toxic disulfide exchanger (e.g., cysteine ​​(Cys)) and reacts it with a membrane-forming protein or pegylated membrane-forming protein to form a protein nanocoating layer having a calcification-regulating function. Since the protein nanocoating layer does not generate large amounts of high molecular weight protein aggregates during the process of regulating the calcification process, blockage of dentin pores is avoided, and it has better permeability, allowing it to penetrate into the interior of already exposed dentin pores, reaching a depth of approximately 200 μm. Moreover, by attracting supersaturated calcium ions and phosphate ions in saliva through the action of electrostatic attraction, hydrophobic interactions, van der Waals forces, etc., it regulates the remineralization process and can form a highly stable HAp layer with a certain orientation in a relatively short time.

[0021] (2) The calcification material of this application does not require special processing of proteins in the process of forming the protein coating layer, nor does it require the custom production of expensive amelogenin peptides. The protein coating layer that regulates remineralization proposed in this application is colorless and transparent, easy to prepare, inexpensive, non-toxic, and has excellent biocompatibility. The remineralizing HAp layer has a dense structure, is highly stable, and calcifies to a depth of 200 μm inside the pits and fissures of dentin.

[0022] (3) The film-forming protein or pegylated film-forming protein of the present application and cysteine undergo a phase transition in the oral cavity to form a two-dimensional nano-order protein film on the surface of teeth (solid-liquid interface). Since the film has a large number of functional groups on its surface, supersaturated calcium ions and phosphate ions in saliva can be adsorbed onto the surface of the film by electrostatic attraction, hydrophobic interaction, van der Waals force, etc. to form a nucleation site. By remineralizing the inside of the dentin micropores and the surface of the defective enamel in saliva in the oral cavity to generate a remineralized coating layer, the micropores can be deeply sealed to achieve the purposes of treating tooth hypersensitivity, repairing enamel, preventing dental caries, and sealing pits and fissures. As the calcification time increases, the depth of the seal for the micropores reaches about 500 μm, and the thickness of the enamel remineralization also increases linearly with the calcification time (see Figure 5). The hydroxyapatite formed by surface remineralization has a certain orientation, and its performance is comparable to that of natural enamel.

[0023] (4) The calcification material of the present application can be used to manufacture dentin desensitizers, mouth rinses, multi-purpose toothpastes, pit and fissure sealants, etc. The dentin desensitizers, mouth rinses, multi-purpose toothpastes, and pit and fissure sealants of the present application utilize the phase transition reaction of film-forming protein molecules or pegylated film-forming protein molecules and water-soluble disulfide exchange agents to form a protein nanofilm with a thickness of about 20 - 45 nm that can regulate remineralization by interface-induced self-assembly inside the dentin micropores, on the surface of defective enamel, and inside the pits and fissures. The film is colorless and transparent, has good adhesion, and has the function of reinforcing the hydroxyapatite newly formed by remineralization on the surface of enamel.

[0024] (5) This application is such that the main component used is a protein, which can effectively block small holes, treat hypersensitivity, effectively repair enamel, prevent acid erosion and demineralization of enamel, and prevent the occurrence of dental caries and pit and fissure caries. This application focuses on the depth of the blockage of dentin small holes, the remineralization effect and the blocking effect on the surface of enamel and inside the pit and fissure grooves. Since hypersensitivity occurs when dentin is exposed from small holes and stimulates the tooth nerve, dental caries and pit and fissure caries are initially recognized as demineralization of the surface of enamel. Therefore, deeply blocking the inside of dentin small holes and remineralizing the surface of enamel by epitaxial growth can prevent and repair dental caries before permanent structural damage appears in the tooth. Or, after permanent structural damage appears, it can deeply block the dentin small holes to treat hypersensitivity, repair the enamel defect by remineralization of enamel, and further prevent the progression of dental caries or pit and fissure caries. After using the tooth desensitizer, mouthwash, multi-purpose toothpaste or pit and fissure sealant for a certain period, remineralization occurs on the surface of demineralized enamel and inside the small holes, or it is mineralized on normal teeth to prevent the occurrence of dental caries and suppress dentin hypersensitivity. The contact time of the tooth desensitizer, mouthwash, multi-purpose toothpaste or pit and fissure sealant with the tooth needs to be about 20 seconds to about 2 minutes. During use, a protein two-dimensional nanofilm with adjustable remineralization can be formed in the inside of the small holes, on the surface of enamel and in the depressions of the pit and fissure grooves. Then, remineralization can occur in the inside of the small holes, on the surface of enamel and in the depressions of the pit and fissure grooves in the saliva in the oral cavity to form a HAp layer.

[0025] (6) The tooth desensitizer, mouthwash, multi-purpose toothpaste or pit and fissure sealant manufactured with the calcifying material of this application has a protein as the main component, and the water-soluble disulfide exchange agent is also a non-toxic protein, so it conforms to the FDA's definition of safe substances and has no harm to the human body when used as a Class II medical device and has excellent biocompatibility. Also, other products for tooth desensitization and preventing enamel demineralization can be manufactured with the calcifying material of this application.

Brief Description of the Drawings

[0026] One or more embodiments are illustrated by images shown in the corresponding drawings, and these illustrative descriptions do not constitute a limitation to the embodiments. As used herein, “exemplary” means “example, as an embodiment, or for illustrative purposes.” No embodiment described “exemplary” herein shall necessarily be construed as better or superior to any other embodiment. [Figure 1] This figure shows the nanofilm and deep film formation formed on the enamel surface and inside the pores of dentin, respectively, by the mouthwash of Test Example 1 and the dental desensitizer of Test Example 2 of the present application. Here, a is a figure showing the interatomic forces of a two-dimensional nanofilm formed by bioproteins on the enamel surface after rinsing with the mouthwash corresponding to Example 1, and b is a figure showing a three-dimensional image obtained by a confocal laser microscope of the induction of deep film formation in the pores of dentin by the dental desensitizer corresponding to Example 1. [Figure 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 this application. [Figure 3]This is a scanning electron microscope image showing the newly formed remineralized coating layer on the enamel surface in the mouthwash test described in Test Example 1 of this application. Here, a is the surface of acid-eroded enamel, b is a cross-section of the enamel, c is the energy spectral analysis of blank enamel, d is a figure showing the surface of enamel after 2 weeks of calcification induction in improved artificial saliva using the mouthwash corresponding to Example 1, e is a figure showing a cross-section of enamel after 2 weeks of calcification induction in improved artificial saliva using the mouthwash corresponding to Example 1, f is a figure showing the energy spectral analysis of the surface of enamel after 2 weeks of calcification induction in improved artificial saliva using the mouthwash corresponding to Example 1, g is a figure showing the surface of enamel after 3 days of calcification induction in actual oral saliva using the mouthwash corresponding to Example 1, h is a figure showing a cross-section of enamel after 3 days of calcification induction in actual oral saliva using the mouthwash corresponding to Example 1, and i is a figure showing the energy spectral analysis of the surface of enamel after 3 days of calcification induction in actual oral saliva using the mouthwash corresponding to Example 1. [Figure 4] These are scanning electron microscope images of a blank dentin section and a dentin section coated with the dental desensitizer corresponding to Example 1. Here, a is the surface of the blank dentin, b is the surface of the dentin coated with the dental desensitizer corresponding to Example 1, c is a cross-section of the blank dentin, d is a cross-section of the dentin after 7 days of bio-calcification without the application of the dental desensitizer, e is a cross-section of the dentin after 7 days of bio-calcification with the application of the dental desensitizer corresponding to Example 1 (maximum depth reaching approximately 200 μm), and f is the energy spectral analysis of the remineralized crystals after the application of the dental desensitizer corresponding to Example 1. [Figure 5]The images show scanning electron microscope images of enamel and the change in calcification thickness with respect to calcification time when using the mouthwash corresponding to Comparative Example 1 and Example 1. Here, a is a figure showing the surface of the enamel after using the mouthwash of Comparative Example 1 for two weeks, b is a figure showing the remineralized cross-section of the enamel after using the mouthwash of Comparative Example 1 for two weeks, c is a figure showing the surface of the enamel after using the mouthwash corresponding to Example 1 of the present application for two weeks, d is a figure showing the remineralized cross-section of the enamel after using the mouthwash corresponding to Example 1 of the present application for two weeks, and e is the change in the thickness of the calcified material with respect to calcification time when using the mouthwash corresponding to Example 1 of the present application. [Figure 6] These are scanning electron microscope images of dentin treated with dental desensitizers corresponding to Comparative Example 2 and Example 1. Here, a is a figure showing the surface of dentin after using the desensitizer obtained in Comparative Example 2 for 3 weeks, b is a remineralized cross-section of dentin after using the desensitizer obtained in Comparative Example 2 for 3 weeks, c is a figure showing the surface of dentin after using the dental desensitizer corresponding to Example 1 of this application for 3 weeks, and d is a figure showing a remineralized cross-section of dentin after using the dental desensitizer corresponding to Example 1 of this application for 3 weeks. [Modes for carrying out the invention]

[0027] The following describes the technical solutions related to the embodiments of this application clearly and completely, so as to make the purpose, technical solutions, and advantages of the embodiments of this application clearer. Needless to say, the embodiments described are some, but not all, of the embodiments of this application. All other embodiments obtained by a person skilled in the art without performing inventive work based on the embodiments of this application shall be considered to fall within the scope of protection of this application.

[0028] Furthermore, in order to further illustrate the present application, many specific details are provided in the following specific embodiments. It will be understood by those skilled in the art that the present application can be implemented in the same manner even without some of these specific details. In some embodiments, in order to highlight the spirit of the present application, details of raw materials, elements, methods, means, etc., which are well known to those skilled in the art, are not described in detail.

[0029] Unless otherwise explicitly stated, throughout the specification and claims, the term “includes” or its variations “incorporate” or “contain” shall be understood to include the elements or components described and not to exclude other elements or components.

[0030] (Example 1) A calcification material for treating hypersensitivity and promoting epitaxial growth of enamel was obtained by uniformly mixing 70 mg of lysozyme, 65 mg of cysteine ​​(Cys), and 20 mg of sodium bicarbonate.

[0031] (Example 2) A calcification material for treating hypersensitivity and promoting enamel epitaxial growth was obtained by uniformly mixing 30 mg of lysozyme, 46 mg of cysteine ​​(Cys), and 15.2 mg of sodium bicarbonate.

[0032] (Example 3) A calcification material for treating hypersensitivity and promoting enamel epitaxial growth was obtained by uniformly mixing 40 mg of lysozyme, 20.8 mg of glutathione (GSH), and 6.2 mg of sodium bicarbonate.

[0033] (Example 4) A calcification material for treating hypersensitivity and promoting enamel epitaxial growth was obtained by uniformly mixing 52 mg of lysozyme, 65 mg of glutathione (GSH), and 18.2 mg of sodium bicarbonate.

[0034] (Example 5) A calcification material for treating hypersensitivity and promoting enamel epitaxial growth was obtained by homogeneously mixing 72 mg of pegylated lysozyme, 67 mg of cysteine ​​(Cys), and 20.6 mg of sodium bicarbonate.

[0035] (Example 6) A calcification material for treating hypersensitivity and promoting enamel epitaxial growth was obtained by homogeneously mixing 62 mg of pegylated lysozyme, 50 mg of glutathione (GSH), and 16.2 mg of sodium bicarbonate.

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

[0037] (Example 8) The mouthwash of this example consists of each of the calcification materials obtained in Examples 1 to 6 above, and 10 mM HEPES buffer. Before use, 20 mg of the calcification material is added to 240 mL of 10 mM HEPES buffer, 6 mg of sodium saccharin and 4 mg of sorbose are 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 example consists of each of the calcification materials obtained in Examples 1 to 6 above, and a 10 mM HEPES buffer. Before use, 35 mg of the calcification material and 120 mL of 10 mM HEPES buffer are uniformly mixed to obtain a dental desensitizer for treating hypersensitivity and promoting enamel epitaxial growth.

[0039] (Example 10) The dental desensitizer of this example consists of each of the calcification materials obtained in Examples 1 to 6 above, and a 10 mM HEPES buffer. When using, 52 mg of the calcification material and 152 mL of 10 mM HEPES buffer are uniformly mixed to obtain a dental desensitizer for treating hypersensitivity and promoting enamel epitaxial growth.

[0040] (Example 11) The pit and fissure sealing material of this example consists of each of the calcification materials obtained in Examples 1 to 6 above, and 10 mM HEPES buffer. When used, 42 mg of the calcification material and 25 mL of 10 mM HEPES buffer are uniformly mixed to obtain a pit and fissure sealing material for treating hypersensitivity and promoting enamel epitaxial growth.

[0041] (Example 12) The pit and fissure sealing material of this example consists of each of the calcification materials obtained in Examples 1 to 6 above, and 10 mM HEPES buffer. When used, 26.2 mg of the calcification material and 30.2 mL of 10 mM HEPES buffer are uniformly mixed to obtain a pit and fissure sealing material for treating hypersensitivity and promoting enamel epitaxial growth.

[0042] (Example 13) The pit and fissure sealing material of this embodiment consists of the calcification material obtained in Examples 1 to 6 above and a toothpaste additive. When using, 12 mg of the calcification material and 20 mg of the toothpaste additive are uniformly mixed to obtain a multipurpose toothpaste for treating hypersensitivity and promoting enamel epitaxial growth.

[0043] (Example 14) The pit and fissure sealing material of this embodiment consists of the calcification material obtained in Examples 1 to 6 above and a toothpaste additive. When used, 45 mg of the calcification material and 100 mg of the toothpaste additive are uniformly mixed to obtain a multipurpose toothpaste for treating hypersensitivity and promoting enamel epitaxial growth.

[0044] In the above examples, lysozyme or pegylated lysozyme is used as: 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, y The pegylated membrane-forming protein may be replaced with one or more of the following: gynosodium serum, fetal bovine serum, mouse serum, immunoglobulin, milk protein, ovalbumin, concanavalin, fish skin collagen, superoxide dismutase, pancrelipase, laccase, histone, collagenase, cellulase, gluten, mucin, transglutaminase, or β-galactosidase. Alternatively, the pegylated membrane-forming protein may be replaced with pegylated lysozyme, pegylated bovine serum albumin, pegylated human serum albumin, or pegylated whey-derived albumin. Bumin, 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 transfer It contains one or more of the following: phosphorus, pegylated fibrinogen, pegylated goat serum, pegylated fetal bovine serum, pegylated mouse serum, pegylated immunoglobulin, pegylated milk protein, 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.

[0045] (Comparative Example 1) The calcification material for this comparative example was obtained by uniformly mixing 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.

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

[0047] (Comparative Example 2) The desensitizer for this comparative example was obtained by uniformly mixing 40 mg of pegylated lysozyme, 10 mg of calcium chloride, 10 mg of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and 60 mg of sodium bicarbonate.

[0048] 60 mg of the calcification 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] To demonstrate the beneficial effects of the present invention, the inventors conducted performance tests on the mouthwash obtained in Example 8 for treating hypersensitivity and promoting enamel epitaxial growth, and the tests were specifically as follows: (Test Example 1) Human third molar samples free from caries were collected (tooth samples were provided by the Hospital of Stomatology, Air Force Medical University and the Hospital of Stomatology, Tianjin Medical University, and this research was approved by the medical research ethics committees of the aforementioned companies). After washing, the enamel was treated with an SYJ160 low-speed microtome and 200-6000 mesh sandpaper to create 6mm x 6mm x 1.5mm enamel sections. The polished enamel sections were acid-etched with 37% (wt%) phosphoric acid for 30 seconds, rinsed with ultrapure water, subjected to ultrasonic treatment for 15 minutes, dried by nitrogen blowing, and then immersed in the mouthwash obtained in Example 8 for 1 minute to simulate the rinsing process.

[0050] After 1 minute, a two-dimensional nanofilm made of bioproteins was formed on the enamel surface. Here, the test results for the mouthwash in Example 8 (a mouthwash produced using the calcification material of Example 1 and the method of Example 8, hereinafter referred to as the mouthwash corresponding to Example 1) showed a thickness of approximately 30-50 nm, as shown in Figure 1a. The calcification status was confirmed in the improved artificial saliva and actual saliva in the oral cavity. 1) Enamel covered with the protein film was immersed in improved artificial saliva and biocalcified at 37°C, with the improved artificial saliva being replaced once daily. During the calcification process, the enamel was removed from the improved artificial saliva daily, rinsed with ultrapure water, dried by nitrogen spraying, immersed for 1 minute in the mouthwash obtained in Example 8, and then transferred to the improved artificial saliva. This process was repeated twice daily. 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 scanned with an energy spectrometer to analyze its elements, an indentation was made in the enamel using a Knoop hardness tester, and the cross-sectional morphology of the remineralized coating layer of the enamel was observed with a scanning electron microscope. Here, the scanning results of the energy spectrometer when using the mouthwash corresponding to Example 1 are shown in Figures 3d and 3f, and the scanning results of the Knoop hardness tester are shown in Figure 3e.

[0051] As shown in Figure 3, the two-dimensional nanofilm made of bioproteins grows on the enamel by in-situ growth. When biocalcification was simulated by placing the enamel coated with the protein film into improved artificial saliva, well-oriented hydroxyapatite was produced, and the calcium / phosphorus ratio matched that of natural enamel. The newly formed hydroxyapatite through remineralization on the surface of acid-eroded enamel (Figures 3a-c) prevents enamel demineralization and thus prevents dental caries.

[0052] 2) The enamel on which the protein film was growing was immersed in actual oral saliva after centrifugation (the actual saliva was collected from healthy adult subjects, the samples were collected at least 2 hours after eating, and the collection of samples was authorized by the Medical Research Ethics Committee), simulating biocalcification by immersing the enamel on which the film was growing in actual oral saliva. After 3 days, the morphology of the enamel surface was observed, and the results are shown in Figures 3g-i. As is clear from the figures, when the enamel on which the protein film was growing was immersed in actual oral saliva, highly oriented regenerated hydroxyapatite was obtained through remineralization, demonstrating that the protein film can exert a calcification effect even in the salivary environment of the oral cavity and can be used in the oral environment of the human body.

[0053] The inventors further compared scanning electron microscope images of film formation and calcified enamel using the mouthwash of Comparative Example 1 and the mouthwash corresponding to Example 1 using the method described above, and the results are shown in Figure 5. As shown in the results, when using the mouthwash corresponding to Example 1, the formed calcified layer had a dense surface (Figure 5c), and the thickness of the remineralized enamel layer increased linearly with calcification time (Figure 5e), whereas with the mouthwash of Comparative Example 1, the surface of the calcified enamel layer was not dense (Figure 5a), and the thickness of the remineralized enamel layer with the mouthwash of Comparative Example 1 (Figure 5b, indicated by the arrow) was far less than the depth of the calcification with the mouthwash corresponding to Example 1 (Figure 5d, indicated by the arrow).

[0054] (Test Example 2) To demonstrate the beneficial effects of the present invention, the inventors further conducted performance tests on the dental desensitizer obtained in Example 10, and the tests were specifically as follows. Human third molar samples free from caries were collected (tooth samples were provided by the Hospital of Stomatology, Air Force Medical University and the Hospital of Stomatology, Tianjin Medical University, and this research was approved by the medical research ethics committees of the aforementioned companies). After washing, 2 mm thick dentin sections were prepared using a SYJ160 low-speed microtome, polished to 5 mm × 5 mm × 2 mm dentin samples, and these dentin samples were caried using a 17% (wt%) EDTA solution and a 2% (wt%) NaClO solution before being used as test samples.

[0055] The aforementioned samples were immersed in a dental desensitizer solution, left to stand at 37°C for 2 minutes, and then removed. Due to the characteristic that the dental desensitizer can specifically bind to the fluorescent dye thioflavin T (ThT), a three-dimensional imaging test using a confocal laser microscope demonstrated that the dental desensitizer could penetrate into the interior of small pores and form a film. The test results for the dental desensitizer in Example 10 (a dental desensitizer manufactured using the method of Example 10 with the calcification material of Example 1, hereinafter referred to as the dental desensitizer corresponding to Example 1) are shown in Figure 1b.

[0056] Dentin sections coated with the aforementioned dental desensitizer were placed in a 12-well plate, and cultured at 37°C with improved artificial saliva. The improved artificial saliva was replaced daily, and after 7 days of remineralization, the sealing of the pores was observed using a scanning electron microscope. Blank sections without the dental desensitizer were used as a control, and the test results for the dental desensitizer corresponding to Example 1 are shown in Figure 4.

[0057] As is clear from Figure 4e, the inside of the pores in the dentin sections coated with a dental desensitizer and a protein film were sealed by remineralization crystals. However, no crystals appeared inside the pores in the dentin sections of the blank control group (Figure 4d) that were not coated with the dental desensitizer, and the dentin remained exposed. Energy spectral analysis (Figure 4f) proved that the remineralization crystals were hydroxyapatite (HAp). This test example demonstrates that the dental desensitizer proposed in this application can penetrate deeply into the pores and form a protein nano-coating layer with remineralization regulating function. Furthermore, it can induce dentin remineralization in an improved artificial salivary environment, deeply sealing the pores in the dentin and thus treating symptoms of hypersensitivity.

[0058] The inventors further extended the remineralization time (3 weeks) using the method described above and compared scanning electron microscope images of calcified dentin treated with the dental desensitizer of Comparative Example 2 and the dental desensitizer corresponding to Example 1. The results are shown in Figure 6. As the results show, with increasing calcification time, the dental desensitizer corresponding to Example 1 achieved a sealing depth of 200-500 μm for the pores (the distance from the surface of the dentin section in frame d in Figure 6 was approximately 420 μm), while the dental desensitizer of Comparative Example 2 achieved a sealing depth of only about 40 μm for the pores (indicated by the white arrow in b in Figure 6).

[0059] (Test Example 3) To determine the boundary between membrane-forming protein and cysteine-mediated membrane formation, the inventors prepared membrane-forming protein and cysteine ​​solutions at different concentrations and plotted the curves shown in Figure 2. As is clear from this figure, at Lyz and Cys concentrations below the left boundary, complete two-dimensional nanofilms capable of controlling the remineralization process cannot aggregate. Also, as can be seen from this figure, when the protein concentration is 0.2 mg / mL or higher, membrane formation is possible with small amounts of cysteine, but when the protein concentration is lower than 0.2 mg / mL, high concentrations of cysteine ​​are required for membrane formation.

[0060] As can be seen from the above, the remineralizing material used in this application is mainly protein, is non-toxic, has excellent biocompatibility, is easy to prepare and use, and in vitro tests have proven that the crystals produced by remineralization have a stable structure and are comparable to natural enamel or dentin, enabling long-term tooth desensitization and demonstrating excellent therapeutic effects. Excellent therapeutic applications are expected for the remineralizing material used in this application to treat hypersensitivity, repair enamel defects and prevent caries, as well as for products such as dental desensitizers, mouthwashes and toothpastes manufactured with this remineralizing material.

[0061] This invention uses protein lysozyme (or pegylated lysozyme) to form a protein nanofilm through the action of a disulfide exchanger (cysteine ​​or glutathione). This nanofilm can be used to treat hypersensitivity and caries, and to seal pits and fissures, by promoting remineralization of dentin and epitaxial growth of enamel. The remineralizing material of this invention can be easily applied by methods such as immersion or coating to enter the interior of already exposed dentin pores, the surface of carious enamel, and the interior of pits and fissures, forming a single layer of protein nanofilm coating with remineralization-inducing function. Saturated calcium ions and phosphate ions in saliva can be induced to remineralize inside the dentin pores to form an HAp layer, promoting HAp remineralization on the enamel surface and achieving epitaxial growth of enamel, thereby achieving the objectives of treating hypersensitivity, preventing caries, and sealing pits and fissures. In this application, the protein coating formed within the dentin and on the surface of the enamel also possesses antibacterial and stain-resistant properties, playing an important role in maintaining oral hygiene and health. All of the calcification materials mentioned in this application are safe substances certified by the U.S. Food and Drug Administration (FDA) and possess superior biocompatibility, thus enabling a wider range of clinical applications.

[0062] Finally, I would like to add that the above embodiments are merely for illustrating the technical solutions of the present application and are not intended to limit them. Although the present application has been described in detail with reference to the above embodiments, it is still possible to modify the technical solutions described in each of the above embodiments, or to replace some of their technical features with equivalent ones, and it will be understood by those skilled in the art that such modifications or replacements will not cause the essence of the technical solutions in question to deviate from the spirit and scope of the technical solutions in each of the embodiments of the present application. [Industrial applicability]

[0063] This application provides a remineralizing material for treating hypersensitivity and promoting enamel epitaxial growth, comprising, by weight, 5 to 72 parts of film-forming protein or pegylated film-forming protein, 4 to 67 parts of water-soluble disulfide exchanger, and 1 to 21 parts of pH adjuster as raw materials, and its use. Dental desensitizers, mouthwashes, multipurpose toothpastes, or pit and fissure sealants manufactured with the remineralizing material of this application are mainly composed of proteins, and the water-soluble disulfide exchanger is also a non-toxic protein, so they do not harm the human body and have excellent biocompatibility. Furthermore, other products for tooth desensitization and prevention of enamel demineralization can also be manufactured with the remineralizing material of this application.

Claims

1. The raw materials consist of 5 to 72 parts by weight of membrane-forming protein or pegylated membrane-forming protein, 4 to 67 parts of water-soluble disulfide exchange agent, and 1 to 21 parts of pH adjuster. The water-soluble disulfide exchange agent is a substance having a mercapto group that can perform a thiol-disulfide exchange reaction with a protein, and the water-soluble disulfide exchange agent is one or more of cysteine ​​and glutathione. A calcification material characterized by the following properties for treating hypersensitivity and promoting enamel epitaxial growth.

2. A calcification material for treating hypersensitivity and promoting enamel epitaxial growth, as described in claim 1, characterized by containing, on a weight basis, 30 to 70 parts by weight of a membrane-forming protein or pegylated membrane-forming protein, 20 to 67 parts by weight of a water-soluble disulfide exchanger, and 4 to 21 parts by weight of a pH adjuster as raw materials.

3. As the membrane-forming protein, one or more of the following can be used: 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 aforementioned 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, and pegy Contains one or more of the following: chymotrypsin, pegylated thyroglobulin, pegylated transferrin, pegylated fibrinogen, pegylated goat serum, pegylated fetal bovine serum, pegylated mouse serum, pegylated immunoglobulin, pegylated milk protein, 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. A calcification material for treating hypersensitivity and promoting enamel epitaxial growth, as described in claim 1.

4. A calcification material for treating hypersensitivity and promoting epitaxial growth of enamel, as described in claim 1, characterized in that the pH adjusting agent 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.

5. A mouthwash for treating dentin hypersensitivity and promoting enamel epitaxial growth, characterized by comprising a calcifying material for treating dentin hypersensitivity and promoting enamel epitaxial growth as described in any one of claims 1 to 4.

6. The mouthwash for treating hypersensitivity and promoting enamel epitaxial growth according to claim 5, further comprising a buffer and additives for diluting the calcifying material.

7. The mouthwash for treating hypersensitivity and promoting enamel epitaxial growth, as described in claim 6, is characterized by being obtained by uniformly mixing a calcifying material, a HEPES buffer, and an additive in a mass:volume:mass ratio of 5-25 mg:10-200 mL:5-15 mg.

8. A dental desensitizer for promoting the epitaxial growth of enamel, A dental desensitizer comprising a HEPES buffer and a calcifying material for treating hypersensitivity and promoting enamel epitaxial growth as described in any one of claims 1 to 4.

9. A toothpaste for treating dentin hypersensitivity and promoting enamel epitaxial growth, characterized by comprising a toothpaste additive and a calcification material for treating dentin hypersensitivity and promoting enamel epitaxial growth as described in any one of claims 1 to 4.

10. A pit and fissure sealing material for treating hypersensitivity and promoting epitaxial growth of enamel, characterized in that it comprises a calcifying material for treating hypersensitivity and promoting epitaxial growth of enamel as described in any one of claims 1 to 4.

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